Chimeric antigen receptor (CAR) constructs and NK cells expressing the CAR constructs

Abstract

The technology described herein is directed to NK cell CAR polypeptides that comprise an intracellular signaling domain, an intracellular costimulatory domain, and / or a transmembrane domain derived from natural killer (NK)-associated polypeptide.In various aspects, the present invention describes polynucleotides, vectors, or cells that express the NK CAR polypeptides, and pharmaceutical compositions that include the NK CAR polypeptides, polynucleotides, vectors, or cells.The present invention also describes methods of using the NK CAR polypeptides, such as for treating various diseases and disorders, such as cancer or infectious diseases. TIFF2024504728000088.tif83150

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 141,621, filed January 26, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing submitted by CE-PCT in ASCII format, which is incorporated by reference in its entirety. This ASCII copy, created on December 29, 2021, is titled 087825-000003WOPT_SL.txt and is 307,704 bytes in size.

[0003] Technical Field The technology described herein relates to natural killer (NK) cell chimeric antigen receptor (CAR) constructs. [Background technology]

[0004] background Adoptive T cell therapy using chimeric antigen receptor (CAR) constructs has been used as a cancer treatment. CAR-T cell therapy can lead to T cell hyperactivity and cytokine release syndrome (CRS). In severe cases, such cytokine storms can lead to organ failure or death. Therefore, there is an urgent need for CAR technologies that can be expressed in immune cell types other than T cells. NK cells are functionally similar to CD8+ cytotoxic T cells and are considered an alternative to T cells for cytotoxic killing of tumor cells. Compared with CAR-T cells, CAR-NK technology offers significant advantages, including a lower incidence of CRS or neurotoxicity and the feasibility of off-the-shelf manufacturing. Currently, most CAR-NK cell studies use CAR constructs designed for CAR-T cells that are not optimized for NK cell activation. Therefore, there is an urgent need to develop CAR constructs for the specific activation of NK cells so that enhanced antitumor activity can be achieved. Summary of the Invention

[0005] overview This technology relates to chimeric antigen receptor (CAR) constructs that can be expressed in natural killer (NK) cells and their use in the treatment of cancer and infectious diseases. Described herein are CAR constructs that can be used for NK cell engineering in methods of modifying NK cells to express the CAR constructs and using CAR-modified NK cells as therapeutic agents. Generally, NK CAR constructs comprise an extracellular domain containing an extracellular binding domain (i.e., an antigen recognition region), a transmembrane domain (TD), and an intracellular domain containing one or two signaling and / or costimulatory domains. This technology provides CAR constructs with transmembrane and / or intracellular domains that are different from those used in CAR-T therapy and are adapted for use in modifying NK cells, for example, to target NK cells to cells expressing a given marker or tumor antigen. NK cells modified with the CAR constructs described herein have more potent NK cell activation and cytotoxic activity than those currently used in CAR-T therapy. Thus, this technology provides CAR constructs that induce high levels of NK cell proliferation, activation, cytokine secretion, and cytolytic activity.

[0006] In one aspect, described herein is a chimeric antigen receptor (CAR) polypeptide for expression in a natural killer (NK) cell, comprising: (a) an extracellular binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one of: (i) an intracellular signaling domain derived from an NK cell receptor; (ii) an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein; and / or (iii) an intracellular costimulatory domain derived from a costimulatory receptor.

[0007] In some embodiments of any aspect, the polypeptide comprises, from N-terminus to C-terminus, (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain.

[0008] In some embodiments of any aspect, the polypeptide comprises, from C-terminus to N-terminus, (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain.

[0009] In some embodiments of any aspect, the NK cell receptor or NK cell membrane-bound signaling adaptor protein is selected from the group consisting of: (a) natural killer cell receptor 2B4, (b) natural killer-, T-, and B-cell antigen (NTB-A), (c) CD2-Like Receptor Activating Cytotoxic Cell (CRACC), (d) cluster of differentiation 2 (CD2), (e) high-affinity IgE receptor (FcεR1), and (f) CD3-zeta (CD3ζ).

[0010] In some embodiments of any aspect, the NK cell receptor is selected from the group consisting of 2B4, NTB-A, CRACC, and CD2.

[0011] In some embodiments of any aspect, the NK cell receptor is 2B4.

[0012] In some embodiments of any aspect, the NK cell receptor is NTB-A.

[0013] In some embodiments of any aspect, the NK cell receptor is CRACC.

[0014] In some embodiments of any aspect, the NK cell receptor is CD2.

[0015] In some embodiments of any aspect, the NK cell membrane-bound signaling adaptor protein is CD3ζ or FcεR1.

[0016] In some embodiments of any aspect, the intracellular signaling domain from an NK cell receptor or the intracellular signaling domain from an NK cell membrane-bound signaling adaptor protein comprises the amino acid sequence of one of SEQ ID NOs:7-12 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:7-12.

[0017] In some embodiments of any aspect, the costimulatory receptor is 4-1BB and / or IL2 receptor beta (IL2RB).

[0018] In some embodiments of any aspect, the intracellular costimulatory domain from a costimulatory receptor comprises the amino acid sequence of one of SEQ ID NOs:15-16 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:15-16.

[0019] In some embodiments of any aspect, the intracellular domain further comprises at least one self-cleaving peptide.

[0020] In some embodiments of any aspect, the self-cleaving peptide is T2A, P2A, E2A, or F2A.

[0021] In some embodiments of any aspect, the self-cleaving peptide comprises the amino acid sequence of one of SEQ ID NOs:19-20 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:19-20.

[0022] In some embodiments of any aspect, the intracellular domain further comprises a cytokine.

[0023] In some embodiments of any aspect, the cytokine is IL-15 or IL-21.

[0024] In some embodiments of any aspect, the cytokine comprises the amino acid sequence of one of SEQ ID NOs:23-24 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:23-24.

[0025] In some embodiments of any aspect, the intracellular domain further comprises at least one self-cleaving peptide and at least one cytokine.

[0026] In some embodiments of any aspect, the cytokine is adjacent to and distal to the self-cleaving peptide such that the cytokine is separated from the polypeptide by the self-cleaving peptide.

[0027] In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of a native NK cell receptor.

[0028] In some embodiments of any aspect, the transmembrane domain of a native NK cell receptor is selected from the group consisting of natural killer group 2D (NKG2D), natural killer cell P46-related protein (NKp46), and DNAX accessory molecule-1 (DNAM1).

[0029] In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of CD8.

[0030] In some embodiments of any aspect, the transmembrane domain comprises an amino acid sequence of one of SEQ ID NOs: 29-32 or 117-118, or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 29-32 or 117-118.

[0031] In some embodiments of any aspect, the extracellular binding domain is an antibody, an antigen-binding fragment thereof, a F(ab) fragment, a F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb).

[0032] In some embodiments of any aspect, the extracellular binding domain comprises an scFv.

[0033] In some embodiments of any aspect, the extracellular binding domain specifically binds to a tumor-associated antigen.

[0034] In some embodiments of any aspect, the tumor-associated antigen is CD19.

[0035] In some embodiments of any aspect, the tumor-associated antigen is CD33.

[0036] In some embodiments of any aspect, the extracellular binding domain comprises the amino acid sequence of one of SEQ ID NOs:35-36 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:35-36.

[0037] In some embodiments of any aspect, the polypeptide further comprises a signal peptide positioned N-terminal to the extracellular binding domain.

[0038] In some embodiments of any aspect, the signal peptide is a CD8 signal peptide.

[0039] In some embodiments of any aspect, the signal peptide comprises the amino acid sequence of SEQ ID NO:38 or an amino acid sequence that is at least 80% identical to SEQ ID NO:38.

[0040] In some embodiments of any aspect, the polypeptide further comprises a detectable marker distal to the extracellular binding domain.

[0041] In some embodiments of any aspect, the detectable marker is 3xFLAG.

[0042] In some embodiments of any aspect, the detectable marker comprises the amino acid sequence of SEQ ID NO:40 or an amino acid sequence that is at least 80% identical to SEQ ID NO:40.

[0043] In some embodiments of any aspect, the polypeptide further comprises a linker domain distal to the extracellular binding domain and / or proximal to the signal peptide and / or detectable marker.

[0044] In some embodiments of any aspect, the linker comprises the amino acid sequence of SEQ ID NO:42 or an amino acid sequence that is at least 80% identical to SEQ ID NO:42.

[0045] In some embodiments of any aspect, the polypeptide further comprises a spacer domain positioned between the extracellular binding domain and the transmembrane domain.

[0046] In some embodiments of any aspect, the spacer domain comprises a CD8 hinge domain.

[0047] In some embodiments of any aspect, the spacer comprises the amino acid sequence of SEQ ID NO:44 or an amino acid sequence that is at least 80% identical to SEQ ID NO:44.

[0048] In some embodiments of any aspect, the polypeptide comprises the amino acid sequence of one of SEQ ID NOs:80-114 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:80-114.

[0049] In one aspect, described herein is a chimeric antigen receptor (CAR) polypeptide for expression in a natural killer (NK) cell, comprising: (a) an extracellular binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one intracellular signaling domain derived from an NK cell receptor.

[0050] In some embodiments of any aspect, the polypeptide comprises, from N-terminus to C-terminus, (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain.

[0051] In some embodiments of any aspect, the polypeptide comprises, from C-terminus to N-terminus, (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain.

[0052] In some embodiments of any aspect, the NK cell receptor is selected from the group consisting of 2B4, NTB-A, CRACC, and CD2.

[0053] In some embodiments of any aspect, the NK cell receptor is 2B4.

[0054] In some embodiments of any aspect, the NK cell receptor is NTB-A.

[0055] In some embodiments of any aspect, the NK cell receptor is CRACC.

[0056] In some embodiments of any aspect, the NK cell receptor is CD2.

[0057] In some embodiments of any aspect, the intracellular signaling domain from the NK cell receptor comprises the amino acid sequence of one of SEQ ID NOs:7-10 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:7-10.

[0058] In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of a native NK cell receptor.

[0059] In some embodiments of any aspect, the transmembrane domain of a native NK cell receptor is selected from the group consisting of natural killer group 2D (NKG2D), natural killer cell P46-related protein (NKp46), and DNAX accessory molecule-1 (DNAM1).

[0060] In some embodiments of any aspect, the transmembrane domain comprises an amino acid sequence of one of SEQ ID NOs: 29-31 or 117-118, or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 29-31 or 117-118.

[0061] In some embodiments of any aspect, the extracellular binding domain is an antibody, an antigen-binding fragment thereof, a F(ab) fragment, a F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb).

[0062] In some embodiments of any aspect, the extracellular binding domain comprises an scFv.

[0063] In some embodiments of any aspect, the extracellular binding domain specifically binds to a tumor-associated antigen.

[0064] In some embodiments of any aspect, the tumor-associated antigen is CD19.

[0065] In some embodiments of any aspect, the tumor-associated antigen is CD33.

[0066] In some embodiments of any aspect, the extracellular binding domain comprises the amino acid sequence of one of SEQ ID NOs:35-36 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:35-36.

[0067] In some embodiments of any aspect, the polypeptide further comprises a signal peptide positioned N-terminal to the extracellular binding domain.

[0068] In some embodiments of any aspect, the signal peptide is a CD8 signal peptide.

[0069] In some embodiments of any aspect, the signal peptide comprises the amino acid sequence of SEQ ID NO:38 or an amino acid sequence that is at least 80% identical to SEQ ID NO:38.

[0070] In some embodiments of any aspect, the polypeptide further comprises a detectable marker distal to the extracellular binding domain.

[0071] In some embodiments of any aspect, the detectable marker is 3xFLAG.

[0072] In some embodiments of any aspect, the detectable marker comprises the amino acid sequence of SEQ ID NO:40 or an amino acid sequence that is at least 80% identical to SEQ ID NO:40.

[0073] In some embodiments of any aspect, the polypeptide further comprises a linker domain distal to the extracellular binding domain and / or proximal to the signal peptide and / or detectable marker.

[0074] In some embodiments of any aspect, the linker comprises the amino acid sequence of SEQ ID NO:42 or an amino acid sequence that is at least 80% identical to SEQ ID NO:42.

[0075] In some embodiments of any aspect, the polypeptide further comprises a spacer domain positioned between the extracellular binding domain and the transmembrane domain.

[0076] In some embodiments of any aspect, the spacer domain comprises a CD8 hinge domain.

[0077] In some embodiments of any aspect, the spacer comprises the amino acid sequence of SEQ ID NO:44 or an amino acid sequence that is at least 80% identical to SEQ ID NO:44.

[0078] In some embodiments of any aspect, the polypeptide comprises the amino acid sequence of one of SEQ ID NOs:90-105 or SEQ ID NOs:110-114, or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:90-105 or SEQ ID NOs:110-114.

[0079] In one aspect, described herein is a chimeric antigen receptor (CAR) polypeptide for expression in a natural killer (NK) cell, comprising: (a) an extracellular binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one of: (i) an intracellular signaling domain derived from a NK cell membrane-bound signaling adaptor protein and / or (ii) an intracellular costimulatory domain derived from a costimulatory receptor.

[0080] In some embodiments of any aspect, the polypeptide comprises, from N-terminus to C-terminus, (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain.

[0081] In some embodiments of any aspect, the polypeptide comprises, from C-terminus to N-terminus, (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain.

[0082] In some embodiments of any aspect, the NK cell membrane-bound signaling adaptor protein is CD3ζ or FcεR1.

[0083] In some embodiments of any aspect, the intracellular signaling domain from a NK cell membrane-bound signaling adaptor protein comprises the amino acid sequence of one of SEQ ID NOs:11-12, or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:11-12.

[0084] In some embodiments of any aspect, the costimulatory receptor is 4-1BB and / or IL2 receptor beta (IL2RB).

[0085] In some embodiments of any aspect, the intracellular costimulatory domain from a costimulatory receptor comprises the amino acid sequence of one of SEQ ID NOs:15-16 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:15-16.

[0086] In some embodiments of any aspect, the intracellular domain further comprises at least one self-cleaving peptide.

[0087] In some embodiments of any aspect, the self-cleaving peptide is T2A, P2A, E2A, or F2A.

[0088] In some embodiments of any aspect, the self-cleaving peptide comprises the amino acid sequence of one of SEQ ID NOs:19-20 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:19-20.

[0089] In some embodiments of any aspect, the intracellular domain further comprises a cytokine.

[0090] In some embodiments of any aspect, the cytokine is IL-15 or IL-21.

[0091] In some embodiments of any aspect, the cytokine comprises the amino acid sequence of one of SEQ ID NOs:23-24 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:23-24.

[0092] In some embodiments of any aspect, the intracellular domain further comprises at least one self-cleaving peptide and at least one cytokine.

[0093] In some embodiments of any aspect, the cytokine is adjacent to and distal to the self-cleaving peptide such that the cytokine is separated from the polypeptide by the self-cleaving peptide.

[0094] In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of CD8.

[0095] In some embodiments of any aspect, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence that is at least 80% identical to SEQ ID NO:32.

[0096] In some embodiments of any aspect, the extracellular binding domain is an antibody, an antigen-binding fragment thereof, a F(ab) fragment, a F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb).

[0097] In some embodiments of any aspect, the extracellular binding domain comprises an scFv.

[0098] In some embodiments of any aspect, the extracellular binding domain specifically binds to a tumor-associated antigen.

[0099] In some embodiments of any aspect, the tumor-associated antigen is CD19.

[0100] In some embodiments of any aspect, the tumor-associated antigen is CD33.

[0101] In some embodiments of any aspect, the extracellular binding domain comprises the amino acid sequence of one of SEQ ID NOs:35-36 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:35-36.

[0102] In some embodiments of any aspect, the polypeptide further comprises a signal peptide positioned N-terminal to the extracellular binding domain.

[0103] In some embodiments of any aspect, the signal peptide is a CD8 signal peptide.

[0104] In some embodiments of any aspect, the signal peptide comprises the amino acid sequence of SEQ ID NO:38 or an amino acid sequence that is at least 80% identical to SEQ ID NO:38.

[0105] In some embodiments of any aspect, the polypeptide further comprises a detectable marker distal to the extracellular binding domain.

[0106] In some embodiments of any aspect, the detectable marker is 3xFLAG.

[0107] In some embodiments of any aspect, the detectable marker comprises the amino acid sequence of SEQ ID NO:40 or an amino acid sequence that is at least 80% identical to SEQ ID NO:40.

[0108] In some embodiments of any aspect, the polypeptide further comprises a linker domain distal to the extracellular binding domain and / or proximal to the signal peptide and / or detectable marker.

[0109] In some embodiments of any aspect, the linker comprises the amino acid sequence of SEQ ID NO:42 or an amino acid sequence that is at least 80% identical to SEQ ID NO:42.

[0110] In some embodiments of any aspect, the polypeptide further comprises a spacer domain positioned between the extracellular binding domain and the transmembrane domain.

[0111] In some embodiments of any aspect, the spacer domain comprises a CD8 hinge domain.

[0112] In some embodiments of any aspect, the spacer comprises the amino acid sequence of SEQ ID NO:44 or an amino acid sequence that is at least 80% identical to SEQ ID NO:44.

[0113] In some embodiments of any aspect, the polypeptide comprises the amino acid sequence of one of SEQ ID NOs:80-89 or SEQ ID NOs:106-109, or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:80-89 or SEQ ID NOs:106-109.

[0114] In one aspect, described herein are polynucleotides that encode the polypeptides described herein.

[0115] In some embodiments of any aspect, the polynucleotide comprises one of SEQ ID NOs: 45-79 or a nucleic acid sequence that is at least 80% identical to one of SEQ ID NOs: 45-79.

[0116] In one aspect, described herein is a vector comprising a polynucleotide described herein.

[0117] In some embodiments of any aspect, the vector comprises a lentiviral vector.

[0118] In one aspect, described herein is a lentivirus comprising a polypeptide described herein, a polynucleotide described herein, or a vector described herein.

[0119] In one aspect, described herein is a cell or population thereof comprising a polypeptide described herein, a polynucleotide described herein, a vector described herein, or a lentivirus described herein.

[0120] In some embodiments of any aspect, the cell comprises an immune cell.

[0121] In some embodiments of any aspect, the immune cells comprise natural killer (NK) cells.

[0122] In one aspect, described herein is a pharmaceutical composition comprising a polypeptide described herein, a polynucleotide described herein, a vector described herein, a lentivirus described herein, or a cell described herein, and a pharmaceutically acceptable carrier.

[0123] In one aspect, described herein is a method of increasing activation of an NK cell or a population thereof, comprising contacting the cell or population thereof with a polypeptide described herein, a polynucleotide described herein, a vector described herein, or a lentivirus described herein.

[0124] In some embodiments of any aspect, contacting the NK cells or population thereof with the polypeptide, polynucleotide, vector, or lentivirus increases the activity of the NK cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% compared to before contact with the polypeptide, polynucleotide, vector, or lentivirus.

[0125] In some embodiments of any aspect, the increased activation of the NK cell or population thereof comprises increased expression of a cytokine or granzyme selected from the group consisting of TNFα, IFNγ, GM-CSF, and granzyme B.

[0126] In some embodiments of any aspect, increased activation of NK cells or a population thereof results in increased specific lysis of target cells.

[0127] In some embodiments of any aspect, the target cell expresses a surface antigen that specifically binds to the extracellular binding domain of the polypeptide.

[0128] In some embodiments of any aspect, the target cell is a cancer cell.

[0129] In some embodiments of any aspect, the target cell is a cell infected with a virus or bacteria.

[0130] In one aspect, described herein is a method of treating a subject in need of a CAR-based therapy, comprising administering to the subject a therapeutically effective amount of a CAR-based therapy selected from the group consisting of a polypeptide described herein, a polynucleotide described herein, a vector described herein, a lentivirus described herein, a cell or population thereof described herein, and a pharmaceutical composition described herein.

[0131] In some embodiments of any aspect, the subject has or has been diagnosed with cancer.

[0132] In some embodiments of any aspect, the subject has or has been diagnosed with adrenal gland cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, gallbladder cancer, gestational trophoblastic disease, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, sarcoma, soft tissue sarcoma, spinal cancer, stomach cancer, testicular cancer, pharyngeal cancer, tumor, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0133] In some embodiments of any aspect, administration of the CAR-based therapy results in increased specific lysis of cancer cells targeted by the CAR.

[0134] In some embodiments of any aspect, the subject has or has been diagnosed with an infectious disease.

[0135] In some embodiments of any aspect, the infectious disease is a viral disease or a bacterial disease.

[0136] In some embodiments of any aspect, administration of the CAR-based therapy results in increased specific lysis of infected cells targeted by the CAR.

[0137] In one aspect, described herein is a method of producing a therapeutic composition, the method comprising introducing a nucleic acid encoding a polypeptide described herein, a polynucleotide described herein, a vector described herein, or a lentivirus described herein into an NK cell under conditions that allow expression of the polypeptide in the cell.

[0138] In some embodiments of any aspect, NK cells are removed from a subject in need of the therapeutic composition prior to introduction of the nucleic acid, polynucleotide, vector, or lentivirus.

[0139] In some embodiments of any aspect, after introduction of the nucleic acid, polynucleotide, vector, or lentivirus, the NK cells are returned to the subject. [Brief explanation of the drawings]

[0140] [Figure 1] Figure 1 is a line graph depicting the cytotoxicity of CD19 NK CAR against the CD19-expressing A549 cell line. [Figure 2] Figure 2 is a bar graph depicting the cytotoxicity of CD19 NK CAR against the Raji cell line (calcein-AM assay). [Figure 3] Figure 3 is a bar graph depicting the cytotoxicity of CD19 NK CAR against the Raji cell line (calcein-AM assay). [Figure 4A] Figure 4A is a line graph depicting the cytotoxicity of CD19 NK CAR against Nalm6-GFP-luciferase cell line (bioluminescence assay). [Figure 4B] Figure 4B is a line graph depicting the cytotoxicity of NK CAR against Nalm6-GFP-luciferase cell line (bioluminescence assay). [Figure 5]Figure 5 is a bar graph depicting the cytotoxicity of CD19 NK CAR against the Raji cell line (calcein-AM assay). [Figure 6A] Figure 6A is a line graph depicting the cytotoxicity of CD19 NK CAR against Nalm6-GFP-luciferase cell line (bioluminescence assay). [Figure 6B] Figure 6B is a line graph depicting the cytotoxicity of NK CAR against Nalm6-GFP-luciferase cell line (bioluminescence assay). [Figure 7] Figure 7 is a bar graph depicting the cytotoxicity of CD19 NK CAR against the Raji cell line (calcein-AM assay). [Figure 8] Figure 8 is a line graph depicting the cytotoxicity of CD19 NK CAR against Nalm6-GFP-luciferase cell line (bioluminescence assay). [Figure 9] Figure 9 is a bar graph depicting the cytotoxicity of CD19 NK CAR against the Raji cell line (Calcein-AM assay). [Figure 10] Figure 10 is a line graph depicting the cytotoxicity of CD19 NK CAR against Nalm6-GFP-luciferase cell line (bioluminescence assay). [Figure 11] FIG. 11 is a bar graph depicting the release of the cytokine TNF-alpha by various CD19 NK CAR cells. [Figure 12] Figure 12 is a bar graph depicting the release of the cytokine IFN-gamma by various CD19 NK CAR cells. [Figure 13] Figure 13 is a bar graph depicting the release of the cytokine GM-CSF by various CD19 NK CAR cells. [Figure 14] FIG. 14 is a bar graph depicting the release of Granzyme B by various CD19 NK CAR cells. [Figure 15] FIG. 15 is a schematic representation of Group I and Group II CD19 NK CAR constructs. [Figure 16] FIG. 16 is a schematic representation of Group III and Group IV CD19 NK CAR constructs. [Figure 17] FIG. 17 is a schematic diagram of the NK CAR construct. [Figure 18] Figure 18 is a schematic diagram of the CD33 NK CAR construct. [Figure 19] Figure 19 is a bar graph depicting the cytotoxicity of CD33 NK CAR against the U937 cell line. [Figure 20A] Figures 20A-20E are a series of 24 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:1. [Figure 20B] Figures 20A-20E are a series of 24 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:1. [Figure 20C] Figures 20A-20E are a series of 24 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:1. [Figure 20D] Figures 20A-20E are a series of 24 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:1. [Figure 20E] Figures 20A-20E are a series of 24 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:1. [Figure 21A] Figures 21A-21D are a series of 24 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:4. [Figure 21B] Figures 21A-21D are a series of 24 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:4. [Figure 21C] Figures 21A-21D are a series of 24 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:4. [Figure 21D]Figures 21A-21D are a series of 24 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:4. [Figure 22A] Figures 22A-22E are a series of 48 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:1. [Figure 22B] Figures 22A-22E are a series of 48 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:1. [Figure 22C] Figures 22A-22E are a series of 48 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:1. [Figure 22D] Figures 22A-22E are a series of 48 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:1. [Figure 22E] Figures 22A-22E are a series of 48 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:1. [Figure 23A] Figures 23A-23D are a series of 48 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:4. [Figure 23B] Figures 23A-23D are a series of 48 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:4. [Figure 23C] Figures 23A-23D are a series of 48 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:4. [Figure 23D] Figures 23A-23D are a series of 48 hour FACS plots of CD33 NK CAR cells at an effector to target ratio of 1:4. [Figure 24]Figure 24A is a bar graph summarizing 24- and 48-hour FACS data of CD33 NK CAR cells at an effector-to-target ratio of 1:1. Figure 24B is a bar graph summarizing 24- and 48-hour FACS data of CD33 NK CAR cells at an effector-to-target ratio of 1:4. DETAILED DESCRIPTION OF THE INVENTION

[0141] Detailed Description This technology relates to chimeric antigen receptor (CAR) constructs that can be expressed in natural killer (NK) cells and their use to engineer or modify NK cells to target cancer cells or pathogen-infected cells that express a given cell surface marker or tumor antigen. While some CAR constructs designed, for example, to target T cells to cells expressing a given marker or tumor antigen, can function to target NK cells to the same target cells, the efficiency of such activity is low given the differences in intracellular signaling between NK cells and T cells. Therefore, CAR constructs designed for function in NK cells can provide better results. Herein, we describe CAR constructs that can be used to engineer NK cells to target and kill cells expressing a given cell surface marker or tumor antigen. Below, we describe compositions, methods, and considerations related to the engineering, preparation, and use of CAR-NKs to treat cancer and infectious diseases.

[0142] Thus, the technology described herein is directed to NK cell CAR polypeptides that include an intracellular signaling domain, an intracellular costimulatory domain, and / or a transmembrane domain derived from a natural killer (NK)-associated polypeptide. In various aspects, the present invention describes polynucleotides, vectors, or cells that express such NK CAR polypeptides, and pharmaceutical compositions that include such NK CAR polypeptides, polynucleotides, vectors, or cells. The present invention also describes methods of using such NK CAR polypeptides, for example, for treating various diseases and disorders, such as cancer or infectious diseases.

[0143] Polypeptides In various aspects, the present specification describes NK CAR polypeptides, which comprise at least one of an extracellular binding domain, a transmembrane domain, an intracellular signaling domain, an intracellular costimulatory domain, a detectable marker, a self-cleaving peptide, and / or a cytokine, or any combination thereof. The specific CARs described herein should not be construed as limiting, but rather are illustrative examples of the application of the principles and techniques described. In various aspects, the present specification describes NK CAR polypeptides, which comprise an extracellular binding domain, a transmembrane domain, and at least one intracellular signaling domain. For example, the following combinations are contemplated herein (see, for example, Table 7):

[0144] (Table 7) Exemplary combinations of domains in NK-CAR polypeptides. "ED" indicates an extracellular domain (which itself may comprise an extracellular binding domain, a signal peptide, a linker domain, a detectable marker, and / or a spacer domain). "TM" indicates a transmembrane domain. "IS" indicates an intracellular signaling domain. "IC" indicates an intracellular costimulatory domain. "DM" indicates a detectable marker. "SP" indicates a self-cleaving peptide. "Cy" indicates a cytokine. TIFF2024504728000002.tif192162TIFF2024504728000003.tif150162

[0145] In some embodiments of any aspect, the NK-CAR polypeptide can comprise an extracellular binding domain, a signal peptide, a linker domain, a spacer domain, a transmembrane domain, an intracellular signaling domain, an intracellular costimulatory domain, a detectable marker, a self-cleaving peptide, and / or a cytokine, as described in further detail herein.For a discussion of the various polypeptides described herein, see U.S. Patent No. 10,640,570 B2; Li et al., 2018, Cell Stem Cell 23:1-12; Bryceson et al., 2006; Blood 107:159-166; Claus et al., 2008, Frontiers in Bioscience 13:956-965.The contents of each of these documents are incorporated herein by reference in their entirety.Exemplary NK-CAR polypeptides are described in further detail herein.

[0146] Intracellular domain In various aspects, the present specification describes NK CAR polypeptides that comprise at least one intracellular domain.As described in more detail herein, such intracellular domains can include, but are not limited to, an intracellular signaling domain, an intracellular costimulatory domain, a self-cleaving peptide, a cytokine and a detectable marker.NK CAR polypeptides can include any combination of an intracellular signaling domain, an intracellular costimulatory domain, a self-cleaving peptide, a cytokine and a detectable marker, as exemplified in Table 7.

[0147] In some embodiments of any aspect, the CAR polypeptide comprises one, two, three, four, five, or more intracellular domains, e.g., selected from at least one intracellular signaling domain, at least one intracellular costimulatory domain, at least one self-cleaving peptide, at least one cytokine, and / or at least one detectable marker. In some embodiments of any aspect, the CAR polypeptide comprises one intracellular domain. In some embodiments of any aspect, the CAR polypeptide comprises two intracellular domains. In some embodiments of any aspect, the CAR polypeptide comprises three intracellular domains. In some embodiments of any aspect, the CAR polypeptide comprises four intracellular domains. In some embodiments of any aspect, the CAR polypeptide comprises five intracellular domains. In some embodiments of any aspect, the CAR polypeptide comprises six intracellular domains. In embodiments comprising multiple intracellular domains, the multiple intracellular domains can be different individual intracellular domains, or multiple copies of the same intracellular domain, or a combination thereof.

[0148] Non-limiting examples of intracellular domains for use in the NK CAR polypeptides described herein include (a) an intracellular signaling domain from an NK cell receptor, (b) an intracellular signaling domain from an NK cell membrane-bound signaling adaptor protein, and / or (c) an intracellular costimulatory domain from a costimulatory receptor.

[0149] In some embodiments of any aspect, the CAR polypeptide comprises (a) an intracellular signaling domain derived from an NK cell receptor, (b) an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein, and (c) an intracellular costimulatory domain derived from a costimulatory receptor. In some embodiments of any aspect, the CAR polypeptide comprises (a) an intracellular signaling domain derived from an NK cell receptor. In some embodiments of any aspect, the CAR polypeptide comprises (b) an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein. In some embodiments of any aspect, the CAR polypeptide comprises (c) an intracellular costimulatory domain derived from a costimulatory receptor.

[0150] In some embodiments of any aspect, the CAR polypeptide comprises (a) an intracellular signaling domain derived from an NK cell receptor, and (b) an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein. In some embodiments of any aspect, the CAR polypeptide comprises (a) an intracellular signaling domain derived from an NK cell receptor, and (c) an intracellular costimulatory domain derived from a costimulatory receptor. In some embodiments of any aspect, the CAR polypeptide comprises (b) an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein, and (c) an intracellular costimulatory domain derived from a costimulatory receptor.

[0151] Intracellular signaling domains The intracellular signaling domain of the NK CAR polypeptide described herein is responsible for activating at least one of the normal effector functions of an immune cell incorporating the CAR. The term "effector function" refers to the specialized function of a cell. For example, the effector function of a natural killer (NK) cell can be cytolytic activity. Thus, the term "intracellular signaling domain" refers to the portion of a polypeptide that transmits an effector function signal (e.g., from an extracellular binding domain that specifically binds to a cognate antigen) to instruct the cell to perform its specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, the truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term "intracellular signaling domain" is intended to encompass any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0152] In some embodiments of any aspect, the CAR polypeptide comprises one, two, three, four, five, or more intracellular signaling domains. In some embodiments of any aspect, the CAR polypeptide comprises three intracellular signaling domains. In embodiments comprising multiple intracellular signaling domains, the multiple intracellular signaling domains can be different individual intracellular signaling domains, or multiple copies of the same intracellular signaling domain, or a combination thereof.

[0153] In some embodiments of any aspect, the CAR polypeptide comprises an intracellular signaling domain derived from an NK cell receptor and / or an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein. In some embodiments of any aspect, the NK cell receptor or NK cell membrane-bound signaling adaptor protein (e.g., from which the at least one intracellular signaling domain is derived) is selected from the group consisting of natural killer cell receptor 2B4, natural killer-T-cell-B-cell antigen (NTB-A), cytotoxic cell-activating CD2-like receptor (CRACC), cluster of differentiation 2 (CD2), high-affinity IgE receptor (FcεR1), and CD3-zeta (CD3ζ). In some embodiments of any aspect, the intracellular signaling domain derived from the NK cell receptor or NK cell membrane-bound signaling adaptor protein comprises an immunoreceptor tyrosine-based activation motif (ITAM), a conserved sequence of four amino acids repeated twice that can initiate various signaling pathways and subsequent NK cell activation. In some embodiments of any aspect, the intracellular signaling domain from the NK cell receptor or from the NK cell membrane-bound signaling adaptor protein comprises an immunoreceptor tyrosine-based switch motif (ITSM), which can lead to activation or inhibition depending on the type of adaptor protein to which it binds.

[0154] In some embodiments of any aspect, the intracellular signaling domain of a CAR polypeptide described herein comprises one of SEQ ID NOs:7-12, or an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:7-12, and maintains the same function (e.g., intracellular signaling upon activation of the CAR polypeptide by binding to a cognate antigen). In some embodiments of any aspect, the intracellular signaling domain of a CAR polypeptide described herein comprises one of SEQ ID NOs:7-12, or an amino acid sequence at least 95% identical to the sequence of one of SEQ ID NOs:7-12, and maintains the same function.

[0155] In some embodiments of any aspect, the intracellular signaling domain of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 1-6, or a nucleic acid sequence comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-6, and maintaining the same function, or a nucleic acid sequence comprising a codon-optimized version of one of SEQ ID NOs: 1-6. In some embodiments of any aspect, the intracellular signaling domain of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 1-6, or a nucleic acid sequence comprising a sequence at least 95% identical to one of SEQ ID NOs: 1-6, and maintaining the same function.

[0156] Multiple cytoplasmic signaling sequences within the cytoplasmic signaling portion of the NK CAR polypeptides described herein can be linked to each other in random order or in a specified order. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A glycine-serine doublet is a particularly preferred linker.

[0157] NK cell receptor Chimeric antigen receptors for modifying NK cells can include variants and / or domains of naturally occurring NK cell receptors. As described herein, NK CAR polypeptides can include an intracellular signaling domain derived from an NK cell receptor. As used herein, the term "NK cell receptor" refers to a polypeptide expressed by an NK cell and including an extracellular domain that interacts with a ligand present outside the cell. NK cell receptors further include a transmembrane domain and at least one intracellular signaling domain, examples of which are described in more detail herein. NK cell receptors can be activating receptors, inhibitory receptors, chemotactic receptors, adhesion receptors, or cytokine receptors, depending on the type of ligand and the type of intracellular signal transduction transmitted by the intracellular signaling domain upon ligand binding. In some embodiments of any aspect, the NK CAR polypeptide described herein includes an NK activating cell receptor. Non-limiting examples of NK activating cell receptors or domains thereof (e.g., intracellular signaling domains) that can be adapted or modified for use in the NK CAR polypeptides described herein include NKp46, CD16, hNKp30, hNKp44, hNKp80, mNKR-P1C, NKG2D, mNKG2D-S, hKIR-S, mAct Ly49, CD94 / NKG2C, CRACC, Ly9, CD84, NTBA, and 2B4. See, for example, Vivier et al. Science 331(6013):44-9(2011), the contents of which are incorporated herein by reference in their entirety. Non-limiting examples of NK activating receptors and their corresponding adaptors are shown in Table 10. In some embodiments, the NK CAR polypeptide comprises the intracellular signaling domain of an activating receptor or adaptor protein listed in Table 10.

[0158] Table 10. Activating receptors and their corresponding adaptor proteins TIFF2024504728000004.tif88128

[0159] In some embodiments of any aspect, the NK cell receptor or a domain thereof (e.g., an intracellular signaling domain) that can be adapted or modified for use in the NK CAR polypeptide described herein is a member of the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors. The SLAM family is closely related to the CD2 family of the immunoglobulin (Ig) superfamily of molecules. Non-limiting examples of SLAM family members include SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10. In some embodiments of any aspect, the NK cell receptor is a member of the CD2 family. In some embodiments of any aspect, the NK cell receptor is selected from the group consisting of 2B4, NTB-A, CRACC, and CD2.

[0160] 1. 2B4 In some embodiments of any aspect, the NK cell receptor or domain thereof (e.g., intracellular signaling domain) that can be adapted or modified for use in the NK CAR polypeptides described herein is the natural killer cell receptor 2B4. The NK cell type I receptor protein 2B4 is also interchangeably referred to as CD244, natural killer cell receptor 2B4 (NKR2B4), signaling lymphocyte-activation molecule family member 4 (SLAMF4), NK cell activation-inducing ligand (NAIL), or Nmrk. 2B4 is a cell surface receptor expressed on natural killer (NK) cells (and some T cells) that mediates non-major histocompatibility complex (MHC)-restricted killing. The natural ligand for 2B4 is CD48, which is expressed on hematopoietic cells. Interaction between NK cells and target cells via the 2B4 receptor regulates the cytolytic activity of NK cells. Alternatively spliced ​​transcript variants encoding various isoforms have been described for the 2B4 gene. The intracellular signaling domain of 2B4 stimulates NK cell cytotoxicity, IFN-gamma production, and degranulation. SLAM family receptors (e.g., 2B4, CRACC, or NTB-A) can contain immunoreceptor tyrosine switch motifs (ITSMs) with the consensus sequence TXYXX-[VI], which have overlapping specificities for activating and inhibitory SH2 domain-containing binding partners. The ITSMs can mediate interactions with the SH2 domains of the adaptor proteins SH2D1A (SAP) and SH2D1B (EAT-2).

[0161] In some embodiments of any aspect, 2B4 comprises SEQ ID NO: 123 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 123 and maintains the same function (e.g., intracellular signaling in response to extracellular ligand binding; ITSM functionality) as SEQ ID NO: 123. In some embodiments of any aspect, the intracellular signaling domain of an NK-CAR described herein comprises the intracellular portion of 2B4 (e.g., SEQ ID NO: 123). In some embodiments of any aspect, the intracellular signaling domain comprises SEQ ID NO:7 (i.e., residues 251-370 of SEQ ID NO:123) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7 and maintains the same function (e.g., intracellular signaling in response to extracellular ligand binding; ITSM functionality) as SEQ ID NO:7.

[0162] The ITSMs of 2B4 include residues 269-274 (ITSM1), 295-300 (ITSM2), 315-320 (ITSM3), and 340-345 (ITSM4) of SEQ ID NO:123, with tyrosine phosphorylation sites at 271, 297, 317, and 342 of SEQ ID NO:123. Thus, in some embodiments of any aspect, an intracellular signaling domain comprising a sequence substantially similar (e.g., at least 70% identical) to the intracellular signaling domain of 2B4 (e.g., residues 251-370 of SEQ ID NO:123, SEQ ID NO:7) does not contain a mutation at residues 269-274, 295-300, 315-320, or 340-345 of SEQ ID NO:123, or does not contain a mutation at residues 271, 297, 317, or 342 of SEQ ID NO:123, or contains a tyrosine at residue 271, 297, 317, or 342 of SEQ ID NO:123.

[0163] SEQ ID NO: 123, Natural killer cell receptor 2B4 isoform 2 precursor [Homo sapiens], NCBI Reference Sequence: NP_001160135.1, 370 amino acids (aa); bold text indicates ITSMs, double underlined text indicates phosphorylation sites. TIFF2024504728000005.tif26161

[0164] SEQ ID NO:1 (polynucleotide) and SEQ ID NO:7 (amino acid) show an exemplary intracellular signaling domain of 2B4 (e.g., residues 251-370 of 2B4; see, e.g., SEQ ID NO:123 for an exemplary full-length 2B4 polypeptide sequence). TIFF2024504728000006.tif43161

[0165] 2. CRACC In some embodiments of any aspect, the NK cell receptor or a domain thereof (e.g., an intracellular signaling domain) that can be adapted or modified for use in the NK CAR polypeptide described herein is a cytotoxic cell-activating CD2-like receptor (CRACC). CRACC is also interchangeably referred to as SLAM family member 7 (SLAMF7), CD319, CS1, membrane protein FOAP-12, CD2 subset 1, protein 19A, novel LY9 (lymphocyte antigen 9)-like protein, or 19A24 protein. CRACC is a cell surface receptor of the CD2 family that triggers NK cell-mediated cytotoxicity. CRACC can associate with the small cytoplasmic adaptor proteins SH2D1A / SAP and / or SH2D1B / EAT-2. CRACC contains an immunoreceptor tyrosine switch motif (ITSM), a tyrosine-based cytoplasmic motif similar to that found in NK cell receptor 2B4.

[0166] In some embodiments of any aspect, CRACC comprises SEQ ID NO: 124 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 124 and maintains the same function (e.g., intracellular signaling that occurs in response to extracellular ligand binding; ITSM functionality) as SEQ ID NO: 124. In some embodiments of any aspect, the intracellular signaling domain of an NK-CAR described herein comprises the intracellular portion of CRACC (e.g., SEQ ID NO: 124). In some embodiments of any aspect, the intracellular signaling domain comprises SEQ ID NO:8 (i.e., residues 248-335 of SEQ ID NO:124) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8 and maintains the same function (e.g., intracellular signaling in response to extracellular ligand binding; ITSM functionality) as SEQ ID NO:8.

[0167] CRACC contains an ITSM-like motif (e.g., residues 282-287 of SEQ ID NO:124) and an ITSM (e.g., residues 302-307 of SEQ ID NO:124), with tyrosine phosphorylation sites at residues 284 and 304 of SEQ ID NO:124. Thus, in some embodiments of any aspect, an intracellular signaling domain comprising a sequence substantially similar (e.g., at least 70% identical) to the intracellular signaling domain of CRACC (e.g., residues 248-335 of SEQ ID NO:124; SEQ ID NO:8) contains no mutations at residues 282-287 or 302-307 of SEQ ID NO:124, or no mutations at residues 284 or 304 of SEQ ID NO:124, or contains a tyrosine at residue 284 or 304 of SEQ ID NO:124.

[0168] SEQ ID NO: 124, CRACC (SLAM family member 7) isoform a precursor [human], NCBI Reference Sequence: NP_067004.3, 335 aa; bold text indicates ITSM-like motif and ITSM, respectively, and double underlined text indicates phosphorylation sites. TIFF2024504728000007.tif25161

[0169] An exemplary CRACC intracellular signaling domain (e.g., residues 248-335 of CRACC; see, e.g., SEQ ID NO:124 for an exemplary full-length 2B4 polypeptide sequence) is shown in SEQ ID NO:2 (polynucleotide) and SEQ ID NO:8 (amino acid). TIFF2024504728000008.tif38161

[0170] 3. NTB-A In some embodiments of any aspect, the NK cell receptor or domain thereof (e.g., intracellular signaling domain) that can be adapted or modified for use in the NK CAR polypeptides described herein is natural killer cell-T cell-B cell antigen (NTB-A). NTB-A is also interchangeably referred to as NK-TB-Ag, SLAM family member 6 (SLAMF6), CD352, Ly108, NTBA, KALI, KALIb, or SF2000. NTB-A is expressed on NK cells as well as T and B lymphocytes. NTB-A undergoes tyrosine phosphorylation and associates with Src homology 2 domain-containing protein (SH2D1A) and SH2 domain-containing phosphatase (SHP). NTB-A can function as a coreceptor in the process of NK cell activation.

[0171] In some embodiments of any aspect, NTB-A comprises SEQ ID NO: 126 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 126 and maintains the same function (e.g., intracellular signaling that occurs in response to extracellular ligand binding; ITSM functionality) as SEQ ID NO: 126. In some embodiments of any aspect, the intracellular signaling domain of an NK-CAR described herein comprises the intracellular portion of NTB-A (e.g., SEQ ID NO: 126). In some embodiments of any aspect, the intracellular signaling domain comprises SEQ ID NO:10 (i.e., residues 248-331 of SEQ ID NO:126) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10 and maintains the same function (e.g., intracellular signaling in response to extracellular ligand binding; ITSM functionality) as SEQ ID NO:10.

[0172] NTB-A contains two ITSMs at residues 282-287 and 306-311 of SEQ ID NO: 126, and has tyrosine phosphorylation sites at residues 284 and 308 of SEQ ID NO: 126. Thus, in some embodiments of any aspect, an intracellular signaling domain comprising a sequence substantially similar (e.g., at least 70% identical) to the intracellular signaling domain of NTB-A (e.g., residues 248-331 of SEQ ID NO: 126; SEQ ID NO: 10) contains no mutations at residues 282-287 or 306-311 of SEQ ID NO: 126, or no mutations at residues 284 or 308 of SEQ ID NO: 126, or contains a tyrosine at residue 284 or 308 of SEQ ID NO: 126.

[0173] SEQ ID NO: 126, NTB-A (SLAM family member 6) isoform 2 precursor [human], NCBI Reference Sequence: NP_443163.1, 331 aa; bold text indicates ITSM, double underlined text indicates phosphorylation sites. TIFF2024504728000009.tif25160

[0174] SEQ ID NO:4 (polynucleotide) and SEQ ID NO:10 (amino acid) show an exemplary NTB-A intracellular signaling domain (e.g., residues 248-331 of NTB-A; see, e.g., SEQ ID NO:126 for an exemplary full-length NTB-A polypeptide sequence). TIFF2024504728000010.tif38161

[0175] 4. CD2 In some embodiments of any aspect, the NK cell receptor or domain thereof (e.g., intracellular signaling domain) that can be adapted or modified for use in the NK CAR polypeptides described herein is cluster of differentiation 2 (CD2). CD2 is also interchangeably referred to as lymphocyte function antigen-2 (LFA-2), P50, sheep erythrocyte receptor (SRBC), T11, Leu-5, erythrocyte receptor, rosette receptor, or LFA-3 receptor. CD2 is expressed by thymocytes, peripheral T cells, NK cells, and a subset of thymic B cells. CD2 interacts with LFA3 (CD58) on antigen-presenting cells. CD2 itself does not have enzymatic activity but transmits signals, in part, through its interaction with CD3 zeta. During spontaneous (e.g., antibody-independent) NK cell cytotoxicity, CD2 recruits CD16 to the NK cell immune synapse. See, e.g., Christian B et al., CD2 Immunobiology. Front Immunol. 2020;11:1090, the contents of which are incorporated herein by reference in their entirety.

[0176] In some embodiments of any aspect, CD2 comprises SEQ ID NO: 125 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 125 and maintains the same function (e.g., intracellular signaling in response to extracellular ligand binding) as SEQ ID NO: 125. In some embodiments of any aspect, the intracellular signaling domain of an NK-CAR described herein comprises the intracellular portion of CD2 (e.g., SEQ ID NO: 125). In some embodiments of any aspect, the intracellular signaling domain comprises SEQ ID NO:9 (i.e., residues 236-251 of SEQ ID NO:125) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9 and maintains the same function as SEQ ID NO:9 (e.g., intracellular signaling in response to extracellular ligand binding).

[0177] SEQ ID NO: 125, T cell surface antigen CD2 isoform 2 precursor [human], NCBI reference sequence: NP_001758.2, 351 aa TIFF2024504728000011.tif26161

[0178] An exemplary CD2 intracellular signaling domain (e.g., residues 236-351 of CD2; see, e.g., SEQ ID NO:125 for an exemplary full-length CD2 polypeptide sequence) is shown in SEQ ID NO:3 (polynucleotide) and SEQ ID NO:9 (amino acid). TIFF2024504728000012.tif43161

[0179] Adaptor proteins In some embodiments of any aspect, the CAR polypeptide comprises an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein. As used herein, the term "NK cell membrane-bound signaling adaptor protein," also referred to interchangeably as "adapter protein," refers to a polypeptide expressed by an NK cell and associated with the cell membrane, which specifically interacts with an NK cell receptor and transmits the receptor's signal via the adaptor's intracellular signaling domain. In some embodiments of any aspect, the NK CAR polypeptide described herein comprises the intracellular region of an adaptor protein associated with an NK-activating cell receptor, which often does not comprise its own intracellular signaling domain. Non-limiting examples of NK adaptor proteins or domains thereof (e.g., intracellular signaling domains) that can be adapted or modified for use in the NK CAR polypeptides described herein include CD3 zeta, FcRγ (which can include FcεRIγ), DAP12, DAP10, SAP, EAT2, and ERT. In some embodiments of any aspect, the NK cell membrane-bound signaling adaptor protein is FcεRI (e.g., FcεRIγ) or CD3 zeta.

[0180] 1. FcεRI In some embodiments of any aspect, the NK cell membrane-bound signaling adaptor protein or domain thereof (e.g., intracellular signaling domain) that can be adapted or modified for use in the NK CAR polypeptides described herein is the high-affinity IgE receptor (FcεRI). FcεRI is also referred to as Fc epsilon RI. FcεRI is a high-affinity receptor for the Fc region of immunoglobulin E (IgE), an antibody isotype involved in allergies and parasite immunity. FcεRI is a tetrameric receptor complex on mast cells and basophils consisting of one alpha chain (FcεRIα—the antibody binding site), one beta chain (FcεRIβ—which amplifies downstream signals), and two gamma chains (FcεRIγ—the site where downstream signals are initiated) connected by two disulfide bridges. In some embodiments of any aspect, the NK cell adaptor protein is FcεRIγ, which is also referred to interchangeably as Fc-epsilon RI-gamma or FCER1G. On human natural killer (NK) cells, FcεRIγ can also associate with FcγRIIIa (CD16a) to form a low affinity receptor for IgG.

[0181] In some embodiments of any aspect, FcεRIγ comprises SEQ ID NO: 127 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 127 and maintains the same function (e.g., intracellular signaling; ITAM functionality) as SEQ ID NO: 127. In some embodiments of any aspect, the intracellular signaling domain of an NK-CAR described herein comprises the intracellular portion of FcεRIγ (e.g., SEQ ID NO: 127). In some embodiments of any aspect, the intracellular signaling domain comprises SEQ ID NO:12 (i.e., residues 45-86 of SEQ ID NO:127) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:12 and maintains the same function (e.g., intracellular signaling; ITAM functionality) as SEQ ID NO:12.

[0182] FcεRIγ contains an ITAM at residues 62-82 of SEQ ID NO:127 and tyrosine phosphorylation sites at residues 65 and 76 of SEQ ID NO:127. Thus, in some embodiments of any aspect, an intracellular signaling domain comprising a sequence substantially similar (e.g., at least 70% identical) to the intracellular signaling domain of FcεRIγ (e.g., residues 45-86 of SEQ ID NO:127; SEQ ID NO:12) contains no mutations at residues 62-82 of SEQ ID NO:127, or no mutations at residues 65 or 76 of SEQ ID NO:127, or contains a tyrosine at residue 65 or 76 of SEQ ID NO:127.

[0183] SEQ ID NO: 127, High-affinity immunoglobulin epsilon receptor subunit gamma precursor [human], NCBI Reference Sequence: NP_004097.1, 86 aa; bold text indicates ITAMs, double underlined text indicates phosphorylation sites. TIFF2024504728000013.tif8160

[0184] An exemplary FceR intracellular signaling domain (e.g., residues 45-86 of FceRI; see, e.g., SEQ ID NO:127 for an exemplary full-length FceRI polypeptide sequence) is shown in SEQ ID NO:6 (polynucleotide) and SEQ ID NO:12 (amino acid). TIFF2024504728000014.tif21160

[0185] 2. CD3-Zeta In some embodiments of any aspect, an NK cell membrane-bound signaling adaptor protein or domain thereof (e.g., intracellular signaling domain) that can be adapted or modified for use in the NK CAR polypeptides described herein is CD3-zeta (CD3ζ). CD3-zeta is also interchangeably referred to as CD247, T cell surface glycoprotein CD3 zeta chain, TCRZ, or IMD25. CD3-zeta contains three ITAMs, which, when phosphorylated, create multiple docking sites for the protein kinase ZAP70, leading to the phosphorylation of ZAP70 and its conversion into a catalytically active enzyme. In NK cells, CD3-zeta can act as an adaptor protein to transmit intracellular signals for the NK activating receptors NKp46, CD16, or hNKp30.

[0186] In some embodiments of any aspect, CD3-zeta comprises SEQ ID NO: 128 or an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 128 and maintains the same function (e.g., intracellular signaling; ITAM functionality) as SEQ ID NO: 128. In some embodiments of any aspect, the intracellular signaling domain of an NK-CAR described herein comprises the intracellular portion of CD3-zeta (e.g., SEQ ID NO: 128). In some embodiments of any aspect, the intracellular signaling domain comprises SEQ ID NO:11 (i.e., residues 52-163 of SEQ ID NO:128) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11 and maintains the same function (e.g., intracellular signaling; ITAM functionality) as SEQ ID NO:11.

[0187] CD3-zeta contains ITAMs at residues 69-89, 107-127, and 138-158 of SEQ ID NO:128, and tyrosine or serine / threonine phosphorylation sites at residues 72, 83, 110, 122, 141, and 152 of SEQ ID NO:128. Thus, in some embodiments of any aspect, an intracellular signaling domain comprising a sequence substantially similar (e.g., at least 70% identical) to the intracellular signaling domain of CD3-zeta (e.g., residues 52-163 of SEQ ID NO:128; SEQ ID NO:11) does not contain a mutation at residues 69-89, 107-127, or 138-158 of SEQ ID NO:128, or does not contain a mutation at residue 72, 83, 110, 122, 141, or 152 of SEQ ID NO:128, or contains a tyrosine at residue 72, 83, 110, 122, 141, or 152 of SEQ ID NO:128.

[0188] SEQ ID NO:128, T cell surface glycoprotein CD3 zeta chain isoform 2 precursor [human], NCBI Reference Sequence: NP_000725.1, 163 aa; bold text indicates ITAMs, double underlined text indicates phosphorylation sites. In some embodiments of any aspect, glutamine-65 (bold-italics) in SEQ ID NO:128 can be changed to lysine-65. TIFF2024504728000015.tif13160

[0189] An exemplary CD3z intracellular signaling domain (e.g., residues 52-163 of CD3z; see, e.g., SEQ ID NO:128 for an exemplary full-length CD3z polypeptide sequence) is shown in SEQ ID NO:5 (polynucleotide) and SEQ ID NO:11 (amino acid). TIFF2024504728000016.tif43160

[0190] Costimulatory domain In some cases, signals generated by NK activating receptors and / or NK adaptor proteins are insufficient for full activation of NK cells, and a secondary or costimulatory signal may also be required. As used herein, the term "costimulatory receptor" refers to a polypeptide expressed by NK cells that includes an extracellular domain that interacts with a costimulatory ligand present outside the cell. Costimulatory receptors typically further include a transmembrane domain and an intracellular signaling domain, examples of which are described in more detail herein. NK cells can be fully activated when signals are transmitted by both the activating receptor and the costimulatory receptor (each of which may transmit its signal via an adaptor protein). In some embodiments of any aspect, a costimulatory receptor or domain thereof (e.g., costimulatory domain) that can be adapted or modified for use in the NK CAR polypeptides described herein is 4-1BB and / or IL2 receptor beta (IL2RB). In some embodiments of any aspect, the CAR polypeptide includes the costimulatory domain of 4-1BB. In some embodiments of any aspect, the CAR polypeptide includes the costimulatory domain of IL-2RB. In some embodiments of any aspect, the CAR polypeptide comprises the costimulatory domains of 4-1BB and IL-2RB.

[0191] In some embodiments of any aspect, the CAR polypeptide comprises one, two, three, four, five, or more costimulatory domains. In some embodiments of any aspect, the CAR polypeptide comprises three costimulatory domains. In those comprising multiple costimulatory domains, the multiple costimulatory domains can be different individual costimulatory domains, or multiple copies of the same costimulatory domain, or a combination thereof.

[0192] In some embodiments of any aspect, the costimulatory domain of a CAR polypeptide described herein comprises one of SEQ ID NOs:15-16 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:15-16 and maintains the same function (e.g., costimulation) as SEQ ID NOs:15-16.

[0193] In some embodiments of any aspect, the costimulatory domain of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 13-14, or a nucleic acid sequence comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 13-14 and maintains the same function, or a nucleic acid sequence comprising a codon-optimized version of one of SEQ ID NOs: 13-14.

[0194] 1. 4-1BB In some embodiments of any aspect, a costimulatory receptor or domain thereof (e.g., costimulatory domain) that can be adapted or modified for use in the NK CAR polypeptides described herein is 4-1BB. 4-1BB is also interchangeably referred to as CD137, tumor necrosis factor receptor superfamily member 9 (TNFRSF9), or receptor induced by lymphocyte activation (ILA). TRAF adaptor proteins have been shown to bind to 4-1BB and transmit signals that lead to the activation of NF-kappa B. 4-1BB expression on human NK cells is associated with phenotypic markers of activation and inflammatory cytokine secretion. The cytoplasmic domain of 4-1BB has been shown to interact with leucine-rich repeat (LRR)-containing protein (LRR-1), which acts as a negative regulator of 4-1BB.

[0195] In some embodiments of any aspect, 4-1BB comprises SEQ ID NO: 129 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 129 and maintains the same function (e.g., costimulation) as SEQ ID NO: 129. In some embodiments of any aspect, the costimulatory domain of an NK-CAR described herein comprises the intracellular portion of 4-1BB (e.g., SEQ ID NO: 129). In some embodiments of any aspect, the costimulatory domain comprises SEQ ID NO:15 (i.e., residues 214-255 of SEQ ID NO:129) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:15 and maintains the same function (e.g., costimulation) as SEQ ID NO:15.

[0196] SEQ ID NO: 129, 4-1BB (Tumor necrosis factor receptor superfamily member 9 precursor) [human], NCBI reference sequence: NP_001552.2, 255 aa. TIFF2024504728000017.tif17161

[0197] An exemplary 4-1BB intracellular costimulatory domain (e.g., residues 214-255 of 4-1BB; see, e.g., SEQ ID NO:129 for an exemplary full-length 4-1BB polypeptide sequence) is shown in SEQ ID NO:13 (polynucleotide) and SEQ ID NO:15 (amino acid). TIFF2024504728000018.tif21159

[0198] 2. IL2RB In some embodiments of any aspect, a costimulatory receptor or domain thereof (e.g., costimulatory domain) that can be adapted or modified for use in the NK CAR polypeptides described herein is interleukin-2 receptor subunit beta (IL-2RB). IL-2RB is also interchangeably referred to as CD122, interleukin-15 receptor subunit beta, P75, or IMD63. The interleukin-2 receptor exists in three forms based on its ability to bind interleukin-2. The low-affinity form is a monomer of the alpha subunit and is not involved in signal transduction. The intermediate-affinity form consists of an alpha / beta subunit heterodimer, while the high-affinity form consists of an alpha / beta / gamma subunit heterotrimer. Both the intermediate-affinity and high-affinity forms of this receptor are involved in receptor-mediated endocytosis and transduction of mitogenic signals from interleukin-2. IL-2 can enhance NK cell responses to infection in vivo and activate NK cells in vitro.

[0199] In some embodiments of any aspect, IL-2RB comprises SEQ ID NO: 130 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 130 and maintains the same function (e.g., costimulation) as SEQ ID NO: 130. In some embodiments of any aspect, the costimulatory domain of an NK-CAR described herein comprises the intracellular portion of IL-2RB (e.g., SEQ ID NO: 130). In some embodiments of any aspect, the intracellular signaling domain comprises SEQ ID NO:16 (i.e., residues 266-337 and 530-551 of SEQ ID NO:130) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:16 and maintains the same function (e.g., costimulation) as SEQ ID NO:16.

[0200] SEQ ID NO:130, Interleukin-2 receptor subunit beta precursor [human], NCBI Reference Sequence: NP_000869.1, 551 aa. TIFF2024504728000019.tif39160

[0201] SEQ ID NO:14 (polynucleotide) and SEQ ID NO:16 (amino acid) show an exemplary IL-2RB intracellular costimulatory domain (e.g., residues 266-337 and 530-551 of IL-2RB; see, e.g., SEQ ID NO:130 for an exemplary full-length IL-2B polypeptide sequence). TIFF2024504728000020.tif38161

[0202] Self-cleaving peptides In some aspects, described herein is an NK CAR polypeptide comprising a self-cleaving peptide. As used herein, the term "self-cleaving peptide" refers to a short amino acid sequence (e.g., approximately 18-22 aa) that can catalyze its own cleavage. In some embodiments of any aspect, the CAR polypeptide described herein comprises an intracellular signaling domain and / or a costimulatory domain physically linked to another polypeptide (e.g., a cytokine) via a self-cleaving peptide domain. The self-cleaving peptide allows the nucleic acid of a first polypeptide (e.g., an NK CAR) and the nucleic acid of a second polypeptide (and / or a third polypeptide, e.g., at least one cytokine) to be present in the same vector, but after translation, the self-cleaving peptide cleaves the translated polypeptide into multiple independent polypeptides.

[0203] In some embodiments of any aspect, a CAR polypeptide described herein comprises one, two, three, four, five, or more self-cleaving peptides. In some embodiments of any aspect, a CAR polypeptide or CAR system comprises one self-cleaving peptide, for example, between a first polypeptide and a second polypeptide. In some embodiments of any aspect, a CAR polypeptide or CAR system comprises two self-cleaving peptides, for example, between a first polypeptide and a second polypeptide and between a second polypeptide and a third polypeptide. In embodiments comprising multiple self-cleaving peptides, the multiple self-cleaving peptides can be different individual self-cleaving peptides, or multiple copies of the same self-cleaving peptide, or a combination thereof.

[0204] In some embodiments of any aspect, the self-cleaving peptide belongs to the 2A peptide family. Non-limiting examples of 2A peptides include P2A, E2A, F2A, and T2A (see, e.g., Table 8). F2A is derived from foot-and-mouth disease virus 18, E2A is derived from equine rhinitis A virus, P2A is derived from porcine teschovirus-1 2A, and T2A is derived from thosea asigna virus 2A. In some embodiments of any aspect, the N-terminal end of the 2A peptide comprises the sequence "GSG" (Gly-Ser-Gly). In some embodiments of any aspect, the N-terminal end of the 2A peptide does not comprise the sequence "GSG" (Gly-Ser-Gly).

[0205] Table 8. Exemplary self-cleaving peptides TIFF2024504728000021.tif32137

[0206] 2A peptide-mediated cleavage begins after protein translation. This cleavage is triggered by the breakdown of the peptide bond between proline (P) and glycine (G) in the C-terminal region of the 2A peptide. The molecular mechanism of 2A peptide-mediated cleavage involves ribosomal "skipping" of glycyl-prolyl peptide bond formation rather than true proteolytic cleavage. Different 2A peptides have different self-cleavage efficiencies, with P2A being the most efficient and F2A being the least efficient. Therefore, up to 50% of the F2A-linked protein can remain in the cell as a fusion protein.

[0207] In some embodiments of any aspect, the self-cleaving peptide of a CAR polypeptide described herein comprises one of SEQ ID NOs: 19-20, 132-134, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs: 19-20, 132-134, and maintains the same function (e.g., self-cleavage) as one of SEQ ID NOs: 19-20, 132-134.

[0208] In some embodiments of any aspect, the self-cleaving peptide of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 17, 18, or is encoded by a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 17 or 18 and maintains the same function as one of SEQ ID NOs: 17 or 18, or is a codon-optimized version of one of SEQ ID NOs: 17 or 18. TIFF2024504728000022.tif25160

[0209] cytokines In some aspects, the present specification describes an NK CAR polypeptide comprising at least one cytokine. As used herein, the term "cytokine" refers to a small protein that mediates and / or regulates a biological function or process or a cellular function or process (e.g., immunity, inflammation, and hematopoiesis). As used herein, the term "cytokine" includes "lymphokines," "chemokines," "monokines," and "interleukins." Examples of cytokines that regulate NK cell function include, but are not limited to, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-γ, GM-CSF, TGF-β, TNF-α, and IFN-α. As used herein, the term "cytokine" also includes cytokine variants containing one or more amino acid mutations in the amino acid sequence of the corresponding wild-type cytokine. In some embodiments of any aspect, the at least one cytokine is linked to the NK CAR polypeptide using at least one self-cleaving peptide described herein. Self-cleaving peptides and cytokines can be used in any combination. The use of a self-cleaving peptide allows the cytokine to be cleaved from the end of the NK CAR polypeptide, and if the cytokine contains a signal peptide, the cytokine can be secreted from the cell and act on the cell that secreted it or nearby cells.

[0210] In some embodiments of any aspect, the CAR polypeptides described herein comprise one, two, three, four, five, or more cytokines. In some embodiments of any aspect, the CAR polypeptide or CAR system comprises one cytokine. In some embodiments of any aspect, the CAR polypeptide or CAR system comprises two cytokines. In embodiments comprising multiple cytokines, the multiple cytokines can be different individual cytokines, or multiple copies of the same cytokine, or a combination thereof.

[0211] In some embodiments of any aspect, the intracellular domain of the NK CAR comprises at least one self-cleaving peptide and at least one cytokine. In some embodiments of any aspect, the intracellular domain of the NK CAR comprises one self-cleaving peptide and one cytokine. In some embodiments of any aspect, the cytokine is adjacent to the self-cleaving peptide and distal to it (i.e., further from the transmembrane domain than the self-cleaving peptide) such that the cytokine is separated from the polypeptide by the self-cleaving peptide. In some embodiments of any aspect, the intracellular domain of the NK CAR comprises two self-cleaving peptides and two cytokines. In some embodiments of any aspect, the first self-cleaving peptide is adjacent to and proximal to the first cytokine (i.e., closer to the transmembrane domain than the first cytokine), and the second self-cleaving peptide is adjacent to and between the first and second cytokines, such that both cytokines are separated from the polypeptide by the self-cleaving peptide.

[0212] In some embodiments of any aspect, the NK CAR polypeptide comprises IL-15 or IL-21. In some embodiments of any aspect, the NK CAR polypeptide comprises IL-15. In some embodiments of any aspect, the NK CAR polypeptide comprises IL-21. In some embodiments of any aspect, the NK CAR polypeptide comprises IL-15 and IL-21. IL-15 is constitutively expressed by multiple cell types and tissues, including monocytes, macrophages, dendritic cells (DCs), keratinocytes, fibroblasts, muscle cells, and neurons. IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 induces the proliferation of natural killer cells. Interleukin-21 (IL-21) is naturally expressed by activated human CD4+ T cells, follicular T cells, and NK T cells. The IL-21 receptor (IL-21R) is expressed on the surface of T, B, and NK cells. IL-21 induces cell division / proliferation of NK cells and cytotoxic T cells. Therefore, the inclusion of IL-15 and / or IL-21 can increase proliferation of NK cells expressing cytokine-containing NK CARs or nearby NK cells.

[0213] In some embodiments of any aspect, the cytokine of a CAR polypeptide described herein comprises one of SEQ ID NOs: 23, 24, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs: 23 or 24, and maintains the same function as one of SEQ ID NOs: 23 or 24 (e.g., promoting NK cell proliferation).

[0214] In some embodiments of any aspect, the cytokine of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising one of SEQ ID NOs:21, 22, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:21 or 22 and maintains the same function, or a nucleic acid sequence that is a codon-optimized version of one of SEQ ID NOs:21 or 22.

[0215] SEQ ID NOs:21 and 23 show the polynucleotide and amino acid sequences of an exemplary IL-15 cytokine (e.g., isoform 1; e.g., interleukin-15 isoform 1 preproprotein [human], see NCBI Reference Sequence: NP_000576.1, 162 aa). TIFF2024504728000023.tif56161

[0216] SEQ ID NOs:22 and 24 show the polynucleotide and amino acid sequences of an exemplary IL-21 cytokine (e.g., isoform 1; e.g., residues 8-162 (155 aa) of IL-21; see e.g., Interleukin-21 isoform 1 precursor [human], NCBI Reference Sequence: NP_068575.1, 162 aa). TIFF2024504728000024.tif56161

[0217] Transmembrane domain In various aspects, the present specification describes NK CAR polypeptides that comprise transmembrane domains.For transmembrane domains, CARs can be designed to comprise transmembrane domains fused to the extracellular binding domain and / or intracellular domain of CAR.In some embodiments of any aspect, CAR polypeptides comprise 1, 2, 3, 4, 5, or more transmembrane domains.In some embodiments of any aspect, each CAR polypeptide comprises one transmembrane domain.In embodiments that comprise multiple transmembrane domains, these multiple transmembrane domains can be different individual transmembrane domains, or multiple copies of the same transmembrane domain, or a combination thereof.

[0218] In some embodiments of any aspect, the transmembrane domain can be derived from either a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In some embodiments of any aspect, the CAR polypeptide described herein comprises at least a transmembrane region of a transmembrane protein selected from the group consisting of NKG2D, NKp46, DNAM, CD8, the alpha chain, beta chain, or zeta chain of the T cell receptor, CD28, CD23 zeta, CD28, 4-1BB, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the NK CAR polypeptide comprises a transmembrane domain of an activating receptor listed in Table 10. Alternatively, the transmembrane domain can be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan, and valine triplet will be found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably greater than 2-10 amino acids, can form the link between the transmembrane domain and the intracellular signaling domain of the CAR. A glycine-serine doublet is a particularly preferred linker.

[0219] In some embodiments of any aspect, the NK CAR comprises a transmembrane domain from the group consisting of natural killer group 2D (NKG2D), natural killer cell P46-related protein (NKp46), DNAX accessory molecule-1 (DNAM1), and CD8. In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of a native NK cell receptor. In some embodiments of any aspect, the transmembrane domain of a native NK cell receptor is selected from the group consisting of natural killer group 2D (NKG2D), natural killer cell P46-related protein (NKp46), and DNAX accessory molecule-1 (DNAM1). In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of CD8. In some embodiments of any aspect, the transmembrane domain further comprises a portion of the adjacent intracellular domain of the same NK cell receptor. As a non-limiting example, the NKp46 or DNAM1 transmembrane domain further comprises a portion of the adjacent intracellular domain of NKP46 or DNAM1, respectively. The inclusion of the intracellular domain can increase the signal transduction of NK CARs, because such receptors can activate NK cells by signal transduction through their own cytoplasmic tails. See, for example, Bryceson et al., 2006, Blood 107:159-166. In some embodiments of any aspect, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, in order to minimize interaction with other members of the receptor complex.

[0220] In some embodiments of any aspect, the transmembrane domain of a CAR polypeptide described herein comprises one of SEQ ID NOs: 29-32 or 117-118, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 29-32 or 117-118 and maintains the same function (e.g., localizes to the cell membrane) as SEQ ID NOs: 29-32 or 117-118.

[0221] In some embodiments of any aspect, the transmembrane domain of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 25-28 or 115-116, or a nucleic acid sequence comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 25-28 or 115-116 and maintains the same function, or a nucleic acid sequence comprising a codon-optimized version of one of SEQ ID NOs: 25-28 or 115-116.

[0222] In some embodiments of any aspect, the transmembrane domain of a CAR polypeptide described herein further comprises an intracellular signaling domain derived from the same protein as the transmembrane domain, and comprises one of SEQ ID NOs:30-31 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:30-31, and that maintains the same function (e.g., cell membrane localization and / or intracellular signaling) as SEQ ID NOs:30-31.

[0223] In some embodiments of any aspect, the transmembrane domain of a CAR polypeptide described herein is encoded by a nucleic acid sequence that further comprises an intracellular signaling domain from the same protein as the transmembrane domain, and that comprises one of SEQ ID NOs:26-27, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:26-27 and maintains the same function, or a codon-optimized version of one of SEQ ID NOs:26-27.

[0224] In some embodiments of any aspect, the transmembrane domain of a CAR polypeptide described herein does not further comprise an intracellular signaling domain from the same protein as the transmembrane domain, but comprises one of SEQ ID NOs: 29, 32, 117, 118, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs: 29 or 32 or 117-118 and maintains the same function (e.g., localizes to the cell membrane) as SEQ ID NO: 29, 32, 117, or 118.

[0225] In some embodiments of any aspect, the transmembrane domain of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 25, 28, 115, 116, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 25, 28, 115, or 116, and maintains the same function, or a sequence that is a codon-optimized version of one of SEQ ID NOs: 25, 28, 115, or 116, without further comprising an intracellular signaling domain from the same protein as the transmembrane domain.

[0226] 1. NKG2D In some embodiments of any aspect, the NK CAR polypeptide comprises at least the transmembrane domain of the NK cell receptor NKG2D. NKG2D is also interchangeably referred to as CD314, killer cell lectin-like receptor subfamily K member 1 (KLRK1), NK cell receptor D, or D12S2489E. NKG2D is a transmembrane protein belonging to the NKG2 family of C-type lectin-like receptors. NKG2D is characterized by a type II membrane orientation (with an extracellular C-terminus) and the presence of a C-type lectin domain. It binds to a diverse family of ligands, including MHC class I chain-related A and B proteins and UL-16 binding proteins, and its ligand-receptor interaction can lead to the activation of NK cells and T cells. NKG2D is an activating NK receptor that signals via the adaptor protein DAP10.

[0227] In some embodiments of any aspect, the NKG2D comprises SEQ ID NO: 135 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 135 and maintains the same function (e.g., cell membrane localization and / or intracellular signaling) as SEQ ID NO: 135. In some embodiments of any aspect, the NK-CAR described herein comprises the transmembrane domain of NKG2D (e.g., SEQ ID NO: 135). In some embodiments of any aspect, the transmembrane domain comprises SEQ ID NO:29 (i.e., residues 52-72 of SEQ ID NO:135) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:29 and maintains the same function as SEQ ID NO:29 (e.g., localizing to the cell membrane).

[0228] SEQ ID NO:135, NKG2-DII type integral membrane protein, UniProtKB-P26718 (NKG2D_HUMAN), 216 aa TIFF2024504728000025.tif17161

[0229] SEQ ID NOs:25 and 29 show the polynucleotide and amino acid sequences of an exemplary NKG2D transmembrane domain (eg, residues 52-72 of NKG2D; see, eg, SEQ ID NO:135 for an exemplary full-length NKG2D polypeptide sequence). TIFF2024504728000026.tif12158

[0230] 2. NKp46 In some embodiments of any aspect, the NK CAR polypeptide comprises at least the transmembrane domain of the NK cell receptor NKp46. NKp46 is also interchangeably referred to as Natural Cytotoxicity Triggering Receptor 1 (NCR1), CD335, and lymphocyte antigen 94 homolog (LY94). NKp46 is a 46-kDa type I membrane glycoprotein expressed on resting and activated NK cells. Its extracellular region contains two C2-type Ig-like domains. The transmembrane domain contains a positively charged amino acid (Arg), which may be involved in stabilizing its association with CD3ζ. Its intracellular region does not contain an immunoreceptor tyrosine-based activation motif (ITAM), but is linked to the cytoplasmic signaling machinery through its association with CD3ζ and the FcεRIγ adaptor protein. Nkp46, together with NKp30 and NKp44, are known as natural cytotoxicity receptors (NCRs), which play a crucial role in killing virus-infected target cells, tumor cells, and MHC class I-unprotected cells.

[0231] In some embodiments of any aspect, Nkp46 comprises SEQ ID NO: 136 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 136 and maintains the same function (e.g., cell membrane localization and / or intracellular signaling) as SEQ ID NO: 136. In some embodiments of any aspect, an NK-CAR described herein comprises the transmembrane domain of NKp46 (e.g., SEQ ID NO: 136). In some embodiments of any aspect, the transmembrane domain comprises SEQ ID NO:117 (i.e., residues 259-279 of SEQ ID NO:136) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:117 and maintains the same function as SEQ ID NO:117 (e.g., localizing to the cell membrane).

[0232] In some embodiments of any aspect, an NK-CAR described herein comprises a transmembrane domain of NKp46 and an intracellular signaling domain of NKp46 (e.g., SEQ ID NO: 136). In some embodiments of any aspect, the transmembrane domain and the intracellular signaling domain comprise SEQ ID NO:30 (i.e., residues 259-304 of SEQ ID NO:136) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:30 and maintains the same function (e.g., cell membrane localization and / or intracellular signaling) as SEQ ID NO:30.

[0233] In some embodiments of any aspect, the NK-CAR described herein comprises the intracellular signaling domain of NKp46 (e.g., SEQ ID NO: 136). In some embodiments of any aspect, the intracellular signaling domain comprises residues 280-304 of SEQ ID NO: 136, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence that includes residues 280-304 of SEQ ID NO: 136, and that maintains the same function (e.g., intracellular signaling) as residues 280-304 of SEQ ID NO: 136.

[0234] SEQ ID NO: 136, Cytotoxicity-induced receptor 1 isoform a precursor [human], NCBI Reference Sequence: NP_004820.2, 304 aa TIFF2024504728000027.tif21161

[0235] SEQ ID NOs:26 and 30 show the polynucleotide and amino acid sequences of an exemplary NKp46 transmembrane and intracellular domain (e.g., residues 259-304 of NKp46; see, e.g., SEQ ID NO:136 for an exemplary full-length NKp46 polypeptide sequence).

[0236] SEQ ID NO:26; bold text indicates sequence encoding an exemplary transmembrane region of NKp46 (e.g., residues 259-279 of SEQ ID NO:136; nucleotides (nt) 1-63 of SEQ ID NO:26; SEQ ID NO:115), and plain text indicates sequence encoding the intracellular signaling domain of NKp46 (see, e.g., residues 280-304 of SEQ ID NO:136). TIFF2024504728000028.tif12160

[0237] SEQ ID NO:30; bold text indicates an exemplary transmembrane region of NKp46 (see, e.g., residues 259-279 of SEQ ID NO:136; amino acids (aa) 1-21 of SEQ ID NO:30; SEQ ID NO:117), and plain text indicates the intracellular signaling domain of NKp46 (e.g., residues 280-304 of SEQ ID NO:136). TIFF2024504728000029.tif4128

[0238] SEQ ID NOs:115 and 117 show the polynucleotide and amino acid sequences of an exemplary NKp46 transmembrane domain (eg, residues 259-279 of NKp46). TIFF2024504728000030.tif17159

[0239] 3. DNAM1 In some embodiments of any aspect, the NK CAR polypeptide comprises at least the transmembrane domain of the NK cell receptor DNAM1. DNAM1 is also interchangeably referred to as DNAX accessory molecule-1, CD226, T lineage-specific activation antigen 1 antigen (TLiSA1), or platelet and T cell activation antigen 1 (PTA1). DNAM1 is a glycoprotein expressed on the surface of NK cells, platelets, monocytes, and a subset of T cells. It is a member of the Ig superfamily containing two V-set Ig-like domains. DNAM1 is involved in cell-cell adhesion, lymphocyte signaling, cytotoxicity, and lymphokine secretion mediated by cytotoxic T lymphocytes (CTLs) and NK cells. DNAM-1 associates with LFA-1 in NK cells, is phosphorylated by PKC, and binds to CD155 and CD112. Antibodies against DNAM-1 inhibit NK cell cytotoxicity against tumor cells.

[0240] In some embodiments of any aspect, DNAM1 comprises SEQ ID NO: 137 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 137 and maintains the same function (e.g., cell membrane localization and / or intracellular signaling) as SEQ ID NO: 137. In some embodiments of any aspect, an NK-CAR described herein comprises the transmembrane domain of DNAM1 (e.g., SEQ ID NO: 137). In some embodiments of any aspect, the transmembrane domain comprises SEQ ID NO:118 (i.e., residues 255-275 of SEQ ID NO:137) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:118 and maintains the same function as SEQ ID NO:118 (e.g., localizing to the cell membrane).

[0241] In some embodiments of any aspect, the NK-CAR described herein comprises a transmembrane domain of DNAM1 and an intracellular signaling domain of DNAM1 (e.g., SEQ ID NO: 137). In some embodiments of any aspect, the transmembrane domain and the intracellular signaling domain comprise SEQ ID NO:31 (i.e., residues 255-336 of SEQ ID NO:137) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:31 and maintains the same function (e.g., cell membrane localization and / or intracellular signaling) as SEQ ID NO:31.

[0242] In some embodiments of any aspect, the NK-CAR described herein comprises the intracellular signaling domain of DNAM1 (e.g., SEQ ID NO: 136). In some embodiments of any aspect, the intracellular signaling domain comprises residues 276-336 of SEQ ID NO: 137, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence that includes residues 276-336 of SEQ ID NO: 137, and maintains the same function (e.g., intracellular signaling) as residues 276-336 of SEQ ID NO: 137.

[0243] SEQ ID NO: 137, CD226 antigen isoform a precursor [human], NCBI reference sequence: NP_001290547.1, 336 aa TIFF2024504728000031.tif25161

[0244] SEQ ID NOs:27 and 31 show the polynucleotide and amino acid sequences of an exemplary DNAM1 transmembrane and intracellular domains (e.g., residues 255-336 of DNAM1; see, e.g., SEQ ID NO:137 for an exemplary full-length DNAM1 polypeptide sequence).

[0245] SEQ ID NO:27; bold text indicates sequence encoding an exemplary transmembrane region of DNAM1 (e.g., residues 255-275 of SEQ ID NO:137; SEQ ID NO:27 nucleotides (nt) 1-63; SEQ ID NO:116), and plain text indicates sequence encoding the intracellular signaling domain of DNAM1 (e.g., residues 276-336 of SEQ ID NO:137). TIFF2024504728000032.tif21161

[0246] SEQ ID NO:31; bold text indicates an exemplary transmembrane region of DNAM1 (e.g., residues 255-275 of SEQ ID NO:137; amino acids (aa) 1-21 of SEQ ID NO:31; SEQ ID NO:118). TIFF2024504728000033.tif8160

[0247] SEQ ID NOs:116 and 118 show the polynucleotide and amino acid sequences of an exemplary DNAM1 transmembrane domain (e.g., residues 255-275 of SEQ ID NO:137). Plain text shows the intracellular signaling domain of DNAM1 (see, e.g., residues 276-336 of SEQ ID NO:137). TIFF2024504728000034.tif12160

[0248] 4. CD8 In some embodiments of any aspect, the NK CAR polypeptide comprises at least the transmembrane domain of CD8. CD8 forms a dimer consisting of a pair of CD8 chains. The most common form of CD8 is composed of a CD8-α chain and a CD8-β chain. Thus, in some embodiments of any aspect, the NK CAR polypeptide comprises at least the transmembrane domain of CD8 alpha. CD8 alpha is also interchangeably referred to as CD8a, T lymphocyte differentiation antigen T8 / Leu-2, or MAL. CD8 is involved in T cell signaling and plays a role in supporting cytotoxic T cell antigen interaction. CD8 is primarily expressed on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells.

[0249] In some embodiments of any aspect, CD8 comprises SEQ ID NO: 138 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 138 and maintains the same function (e.g., cell membrane localization and / or intracellular signaling) as SEQ ID NO: 138. In some embodiments of any aspect, the NK-CAR described herein comprises the transmembrane domain of CD8 (e.g., SEQ ID NO: 138). In some embodiments of any aspect, the transmembrane domain comprises SEQ ID NO:32 (i.e., residues 183-206 of SEQ ID NO:138) or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:32 and maintains the same function as SEQ ID NO:32 (e.g., localizes to the cell membrane).

[0250] SEQ ID NO:138, T cell surface glycoprotein CD8 alpha chain, UniProtKB-P01732 (CD8A_HUMAN), 235 aa TIFF2024504728000035.tif16161

[0251] SEQ ID NOs:28 and 32 show the polynucleotide and amino acid sequences of an exemplary CD8 transmembrane domain (e.g., residues 183-206 of CD8; see, e.g., SEQ ID NO:138 for an exemplary full-length CD8a polypeptide sequence). TIFF2024504728000036.tif17161

[0252] Extracellular domain In some aspects, the present specification describes an NK CAR polypeptide comprising at least one extracellular domain (also referred to herein as an ectodomain).As described in more detail herein, such extracellular domains can include, but are not limited to, an extracellular binding domain, a signal peptide, a linker domain, a detectable marker, and a spacer domain.The NK CAR polypeptide can include any combination of an extracellular binding domain, a signal peptide, a linker domain, a detectable marker, and a spacer domain.For example, the following combinations are contemplated herein (see, for example, Table 8):

[0253] Table 8: Exemplary combinations of extracellular domains in NK-CAR polypeptides. "EB" indicates an extracellular binding domain. "SI" indicates a signal peptide. "LD" indicates a linker domain. "DM" indicates a detectable marker. "SD" indicates a spacer domain. TIFF2024504728000037.tif66164

[0254] In some embodiments of any aspect, the CAR polypeptide comprises one, two, three, four, five, or more extracellular domains. In some embodiments of any aspect, the CAR polypeptide comprises five extracellular domains. In some embodiments of any aspect, the CAR polypeptide comprises four extracellular domains. In embodiments comprising multiple extracellular domains, the multiple extracellular domains can be different individual extracellular domains, or multiple copies of the same extracellular domain, or a combination thereof.

[0255] Extracellular binding domain In some embodiments of any aspect, the NK CAR polypeptides described herein comprise an extracellular binding domain. In some embodiments of any aspect, the extracellular binding domain recognizes and binds to a tumor antigen or a cell surface marker expressed on a tumor cell. In some embodiments of any aspect, the CAR polypeptides described herein comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten extracellular binding domains. In some embodiments of any aspect, the CAR polypeptide or CAR system comprises one extracellular binding domain. In some embodiments of any aspect, the CAR polypeptide comprises two extracellular binding domains. In embodiments comprising multiple extracellular binding domains, the multiple extracellular binding domains can be different individual extracellular binding domains, or multiple copies of the same extracellular binding domain, or a combination thereof.

[0256] In some embodiments of any aspect, the extracellular binding domain is an antibody, an antigen-binding fragment thereof, a F(ab) fragment, a F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments of any aspect, the extracellular binding domain is monovalent, bivalent, or multivalent. In some embodiments of any aspect, the extracellular binding domain comprises a human antibody construct, a humanized antibody construct, or a chimeric antibody construct. In addition, recombinant humanized antibodies can be further optimized for human therapy to reduce potential immunogenicity while maintaining functional activity. In this context, functional activity refers to a polypeptide capable of exhibiting one or more known functional activities associated with the recombinant antibodies or antibody reagents described herein. Such functional activities include, for example, the ability to bind to a target.

[0257] Antibody reagents specific for the targets and / or markers described herein, e.g., tumor antigens, are known in the art. For example, such reagents are readily available commercially. In some embodiments of any aspect, the extracellular binding domain can be an antibody reagent comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs of any one of the antibodies described herein or known in the art. In some embodiments of any aspect, an antibody reagent specific for a target and / or marker described herein (e.g., one that specifically binds to a tumor antigen described herein) can be an antibody reagent comprising six CDRs of any one of the antibodies described herein or known in the art. In some embodiments of any aspect, an antibody reagent specific for a target and / or marker described herein (e.g., one that specifically binds to a tumor antigen described herein) can be an antibody reagent comprising three heavy chain CDRs of any one of the antibodies described herein or known in the art. In some embodiments of any aspect, an antibody reagent specific for a target and / or marker described herein (e.g., one that specifically binds to a tumor antigen described herein) can be an antibody reagent comprising the three light chain CDRs of any one of the antibodies described herein or known in the art. In some embodiments of any aspect, an antibody reagent specific for a target and / or marker described herein (e.g., one that specifically binds to a tumor antigen described herein) can be an antibody reagent comprising the VH and / or VL domains of any one of the antibodies described herein or known in the art. In some embodiments of any aspect, an antibody reagent specific for a target and / or marker described herein (e.g., one that specifically binds to a tumor antigen described herein) can be an antibody reagent comprising the VH and VL domains of any one of the antibodies described herein or known in the art. Such antibody reagents are specifically contemplated for use in the methods and / or compositions described herein.

[0258] As used herein, "antibody variable domain" refers to the portion of the light and heavy chains of an antibody molecule that comprises the amino acid sequences of the complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. With respect to the methods and compositions described herein, the amino acid positions assigned to the CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)). The amino acid numbering of an antibody or antigen-binding fragment also follows the Kabat numbering.

[0259] The terms "antigen-binding fragment" or "antigen-binding domain" are used interchangeably herein and refer to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed by the term "antigen-binding fragment" of a full-length antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments disulfide-bridged at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment consisting of the VH or VL domain (Ward et al., (1989) Nature 341:544-546, which is incorporated herein by reference in its entirety); and (vi) an isolated complementarity-determining region (CDR) that retains specific antigen-binding functionality. The sequences from the light and heavy chains can be provided in N-terminal to C-terminal order, respectively, or in reverse order.

[0260] As used herein, the term "linker" refers to a chemical or peptide structure that covalently links two peptide moieties. For example, the VH and VL domains of an antibody can be linked by a peptide linker to form a VH / VL single-chain antigen-binding domain (e.g., as in an scFv). The length of the linker can be varied to alter the ability of the linked domains to form, for example, intramolecular or intermolecular dimers. For example, diabodies contain a short linker peptide, usually five amino acids, between the VH and VL domains that does not allow the VH and VL domains to pair to form an antigen-binding domain. When two different VH-VL constructs with this short linker configuration are expressed in a single cell, the VH domain of the first VH-VL polypeptide chain can dimerize with the VL domain of the second VH-VL polypeptide chain, and the corresponding VL domain of the first VH-VL polypeptide chain can dimerize with the VH domain of the second VH-VL polypeptide chain, thereby generating a dual-specific construct. In contrast, VH and VL domains on the same polypeptide chain can dimerize to form scFvs when they are separated by a longer peptide linker, most often 15-20 amino acids, non-limiting examples of which are described in further detail herein.

[0261] In some embodiments of any aspect, the extracellular binding domain comprises an scFv. In some embodiments of any aspect, the extracellular binding domain comprises an scFv that specifically binds to a tumor antigen. In some embodiments of any aspect, the extracellular binding domain of a CAR polypeptide described herein specifically binds to any tumor antigen, including those described herein. In some embodiments of any aspect, the extracellular binding domain comprises any known antibody, antigen-binding fragment thereof, F(ab) fragment, F(ab') fragment, single-chain variable fragment (scFv), or single-domain antibody (sdAb) that binds to a tumor antigen or epitope described herein.

[0262] Non-limiting examples of tumor antigens that can be targeted include EphA2, HER2, AXL, GD2, glypican-3, 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor a, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL1 1Ra, IL13Ra2, KDR, lambda, Lewis Y, MCSP, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, survivin, TAG72, TEM1, TEM8, VEGRR2, carcinoembryonic antigen, HMW-MAA, and VEGF receptor. Other exemplary antigens that can be targeted are antigens that are present in the extracellular matrix of tumors or in necrotic areas of tumors, such as, for example, fibronectin, oncofetal variants of tenascin.

[0263] Additional tumor-selective molecules that can be targeted include any membrane protein or biomarker expressed or overexpressed in tumor cells, including, but not limited to, integrins (e.g., integrin αvβ3, α5β1), EGF receptor family (e.g., EGFR2, Erbb2 / HER2 / neu, Erbb3, Erbb4), proteoglycans (e.g., heparan sulfate proteoglycans), disialogangliosides (e.g., GD2, GD3), B7-H3 (also known as CD276), cancer antigen 125 (CA-125), epithelial cell adhesion molecule (EpCA), and the like. M), vascular endothelial growth factor receptor 1 and 2 (VEGFR-1, VEGFR-2), CD52, carcinoembryonic antigen (CEA), tumor-associated glycoproteins (e.g., TAG-72), cluster of differentiation 19 (CD19), CD20, CD22, CD30, CD33, CD40, CD44, CD74, CD152, mucin 1 (MUC1), tumor necrosis factor receptor (e.g., TRAIL-R2), insulin-like growth factor receptor, folate receptor a, transmembrane glycoprotein NMB (GPNMB), CC chemokine receptor (e.g., CCR4), prostate-specific membrane antigen (PSMA), RON (receptor d'origine nantais) receptor, cytotoxic T lymphocyte antigen 4 (CTLA4), and other tumor-specific receptors or antigens.

[0264] Non-limiting examples of tumor antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed fetal antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, and CA15-3. 27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0265] In some embodiments of any aspect, the tumor antigen is a tumor antigen described in International Application PCT / US2015 / 020606 or U.S. Patent Application Publication US20170209492 or US20170335281, the contents of each of which are incorporated by reference in their entirety. In some embodiments, the tumor antigen is chosen from one or more of the following: CD19; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also known as CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDG1cp(1-1)Cer); TNF receptor family member B-cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (Testis Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); nervous cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP);Insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein (bcr-ab1) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Ab1); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl-GM1; sialyl-L1 sLe; ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDG1cp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member -D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex Merger, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;Tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin-8); melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAXS); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2);Lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the tumor antigen is GFRa4 (see, e.g., Spinasanta, "The Endocrine Society's 97th Annual Meeting & Expo: Targeted Therapies in Medullary Thyroid Cancer," Mar. 13, 2015).

[0266] In some embodiments of any aspect, the extracellular binding domain is selected from the group consisting of 20-(74)-(74) (milatuzumab; veltuzumab), 20-2b-2b, 3F8, 74-(20)-(20) (milatuzumab; veltuzumab), 8H9, A33, AB-16B5, abagovomab, abciximab, abituzumab, ABP494 (cetuximab biosimilar), abrilumab, AB T-700, ABT-806, Actimab-A (Actinium Ac-225 lintuzumab), actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG228, AMG820, anatumomab mafenatox, anetumab ravtansineravtansine, anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003 / SIM-BD0801 (sevacizumab), APX005M, arcitumomab, ARX788, ascrinvacumab, aselizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also called tocilizumab), atorolimumab, avelumab, B-701, bapineuzumab, basiliximab, ba Bituximab, BAY1129980, BAY1187982, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, BETALUTIN (177Lu-tetraxetan-tetulomab), bevaciztunab, BEVZ92 (bevacizumab biosimilar), bezlotoxumab, BGB-A317, BHQ880, BI836880, Bl-505, biciromab, bimagnunab, bimekizumab, bivatuzumab mertansinemertansine, BM-8962, blinatumomab, brosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, BREVAREX, briakinumab, brodalumab, brolucizumab, brontiximab, C2-2b-2b, canakinumab, cantuzumab mertansine, cantuzumab vedotin, capra Caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab), CC-90002, CDX-014, CDX-1401, cedelizumab, certolizumab pegol, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14.18, sitatuzumab bogatoxin bogatox), cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, COTARA (Iodine 1-131 Biotin-Derlotuximab), cR6261, crenezumab, DA-3111 (trastuzumab biosimilar), dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, daratumumab ENHANZE (daratumumab), DARLEUKIN, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuximab, depatuximab mafodotin, derlotuximabbiotin), detumomab, DI-B4, dinutuximab, diridavtunab, DKN-01, DMOT4039A, dorlimomab alitoxin aritox, drozittunab, DS-1123, DS-8895, durigotumab, dupilumab, domvalumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efinumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin vedotin), enlimomab pegol, enoblitzumab, enokizumab, enoticumab, encituximab, epitumomabcituxetan), epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivinunab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, FF-21101, FGFR2 antibody-drug conjugate, FIBROMUN, ficlatuzumab, figitumumab, firivumab, framvotumab, fletikumab, fontolizumab, foralumab foralumab, foravirumab, FPAI44, fresolimumab, FS102, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gerilimzumab, gevokizumab, girentuximab, glenbatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3I74998, GSK3359609, guselkumab, Hu14.18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, IGN002, IGN523, igovomab, IMAB362, IMAB362 (claudiximab), imalumab, IMC-CS4, IMC-DII, imciromab, imgatuzumab, IMGN529, IMMU-IO2 (yttrium Y-90 epratuzumab tetraxetan), IMM1J-114, ImmuTune IMP701 antagonist antibody, INCAGN1876, inlacumab, INCSHR1210, indatuximab ravtansine, indusaturnab vedotinvedotin), infliximab, inolimomab, inoturtunab, ozogamicin, intetumumab, ipafricept, IPH4102, ipilimumab, iratumumab, isatuximab, istiratumab, itolizumab, ixekizumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L191L2 / L19TNF, labetuzumab, labetuzumab gobi Tecan, LAG525, lambrolizumab, lampalizumab, L-D0S47, lebrikizumab, lemaresomab, lenzilumab, lerdelimumab, leukotuximab, lexattunumab, libivirumab, rifastuzumab vedotin vedotin, ligelizumab, lilotomab satetlaxetan, lintuzumab, lirilumab, LICZ145, lodelcizumab, lokivetmab, lorvoturtunab mertansine, lucattmumab, lulizumab pegolpegol), lumiliximab, lumretuzumab, LY3164530, mapatumumab, marjetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDT0680, MEDI-3617, MEDI-551 (inebilizumab), MEDI-565, MEDI6469, mepolizumab, metelimumab, MGB453, MGD006 / S80880, MG D007, MGD009, MGD011, milaturtomab, milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitumomab, MK-4166, MM-111, MM-151, MM-302, mogamuliztunab, M0R202, M0R208, MORAb-066, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab butafenatox tafenatox, namilumab, naptumomab estafenatox, narutumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentane, NOV-10, obinutuzumab, ocralizumab, ocrelizumab , odulimomab, ofatumumab, olarattmab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatoxmonatox), oregovomab, orticumab, otelixizumab, otlertuzumab, 0X002 / MEN1309, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pancomab, PankoMab-GEX, panobacumab, palsatuzumab, pascolizumab, pascolizumab, Pasotuxizutnab, pateclizumab, patrittunab, PAT-SC1, PAT-SM6, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, PF-05082566 (utomilumab), PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin vedotin, pintumomab, placurab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, prittunumab, PRO140, PROXINIUM, PSMAADC, kilimumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810 / 5AR439 684, resliztunab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilottunumab, rinucumab, rituximab, RM-1929, R07009789, lobatumumab, rol edumab), romosozumab, lontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, 5AR408701, 5AR566658, sarilumab, SAT012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, seribantumab, setoxaximab mab), sevilumab, SGN-CD19A, SGN-CD19B, SGN-CD33A, SGN-CD70A, SGN-LIVIA, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, sipliztunab, sirukumab, sofituzumab vedotin vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamumab, sulesomab, suvizumab, 5YD985, SYM004 (futuximab and modotuximab), SYM015, TAB08, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, tanibirumab, taplitumomab paptoxpaptox, tarextumab, TB-403, tefibazumab, TELEUKIN, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, TG-1303, TGN1412, thorium-227-epratuzumab conjugate, ticilimumab, tigatuzumab, tildrakizumab, tisotumab vedotin), TNX-650, tocilizumab, toralizumab, tosatoxumab, tositunomab, tobetumab, tralokinumab, trastuzumab, tras Tuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrtunab, TRPH011, TRX518, TSR-042, TTI-200.7, tucotuzumab celmoleukin, tuvilumab, U3-1565, U3-1784, ublituximab, urocuplumab, urelumab, urtoxazumab, ustekinumab, vadastuximab butarilin The antibody or extracellular binding domain thereof (e.g., an antigen-binding domain) is selected from the group consisting of: Talirine, bundeltuzumab vedotin, vanticizumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, VB6-845, vedolizumab, veltuzumab, vepalimomab, besencumab, visilizumab, volociximab, vorseturtunab mafodotin, votumumab, YYB-101, zaluttunumab, zanolimumab, zatuximab, ziralimumab, and zolimonad.

[0267] In some embodiments of any aspect, the extracellular binding domain comprises an anti-CD19 antibody, e.g., an anti-CD19 scFV. CD19 is also interchangeably referred to as B-lymphocyte surface antigen B4 or T-cell surface antigen Leu-12. Because CD19 is a marker for B cells, this protein has been used to diagnose cancers arising from this type of cell, particularly B-cell lymphoma, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). Most B-cell malignancies express normal to high levels of CD19. Non-limiting examples of anti-CD19 antibodies include A3B1, FMC63, FMC63-28Z, and SEQ ID NO:35. See, e.g., U.S. Patent Nos. 10,221,245, 8,906,682, 10,421,810, and 10,639,329, the contents of each of which are incorporated herein by reference in their entirety.

[0268] In some embodiments of any aspect, the extracellular binding domain comprises an anti-CD33 antibody, such as an anti-CD33 scFV. CD33 is also interchangeably referred to as sialic acid-binding Ig-like lectin 3 (SIGLEC3) or Gp67. CD33 is a myeloid-specific sialic acid-binding bioreceptor expressed on approximately 90% of blasts and AML stem cells in acute myeloid leukemia (AML) patients. Non-limiting examples of anti-CD33 antibodies include SEQ ID NO: 36, gemtuzumab, vadastuximab, and 2H12. See, for example, U.S. Patent Nos. 7,557,189, 9,815,901, 10,556,951, and 10,787,514. The contents of each of these documents are incorporated herein by reference in their entirety.

[0269] Depending on the antigen of interest to be targeted, the CAR polypeptides described herein can be engineered to include an appropriate extracellular binding domain (e.g., an antigen-binding portion) specific for the antigen target of interest. For example, if CD19 is the antigen of interest to be targeted, an antibody against CD19 or its extracellular binding domain (e.g., an antigen-binding fragment) can be used as the extracellular binding domain for incorporation into the CAR polypeptides described herein. As another example, if CD33 is the antigen of interest to be targeted, an antibody against CD33 or its extracellular binding domain (e.g., an antigen-binding fragment) can be used as the extracellular binding domain for incorporation into the CAR polypeptides described herein.

[0270] In some embodiments of any aspect, the extracellular binding domain of a CAR polypeptide described herein comprises one of SEQ ID NOs:35-36 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:35-36 and maintains the same function (e.g., tumor antigen binding) as one of SEQ ID NOs:35-36.

[0271] In some embodiments of any aspect, the extracellular binding domain of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising SEQ ID NO:33-34, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NO:33-34 and maintains the same function, or a nucleic acid sequence comprising a codon-optimized version of one of SEQ ID NO:33-34.

[0272] SEQ ID NOs:33 and 35 show the polynucleotide and amino acid sequences of exemplary anti-CD19 scFvs. TIFF2024504728000038.tif78161

[0273] SEQ ID NOs:34 and 36 show the polynucleotide and amino acid sequences of exemplary anti-CD33 scFvs. TIFF2024504728000039.tif77161

[0274] In some embodiments of any aspect, the extracellular binding domain can target an antigen involved in a disease other than cancer (e.g., an infectious disease, etc.). Thus, in some embodiments of any aspect, the extracellular binding domain can target a viral antigen. In some embodiments of any aspect, the extracellular binding domain can target a bacterial antigen. In some embodiments of any aspect, the viral target antigen is a viral surface antigen, e.g., a viral glycoprotein. Non-limiting examples of viral glycoproteins include respiratory syncytial virus (RSV) F protein (e.g., F protein site II); herpesvirus glycoprotein B (gB); hepatitis B virus (HBV) surface antigen (HBsAg); human immunodeficiency virus (HIV) glycoprotein 120 (gp120) (e.g., gp120 CD4 binding site (CD4bs); gp120 third variable region (V3)); influenza virus hemagglutinin (HE; e.g., the HE stalk; e.g., group 1 (G1) or group 2 (G2) HE stalk), and the like.

[0275] In some embodiments of any aspect, the bacterial target antigen is a bacterial surface antigen, such as a bacterial surface protein, lipid, or polysaccharide (i.e., exopolysaccharide). Non-limiting examples of bacterial surface antigens include Staphylococcus protein A; Staphylococcus cell wall teichoic acid; lipopolysaccharide (LPS) (e.g., Pseudomonas aeruginosa serotype O11 LPS); alginate (e.g., Pseudomonas aeruginosa extracellular slime alginate); and Clostridium difficile cell wall polysaccharide 2 (PsII). See, e.g., Wagner et al. Current Opinion in Chemical Engineering 19 (2018): 131-141. In some embodiments of any aspect, the extracellular binding domain can target the antigen using, for example, a biotinylated antigen-specific molecule instead of an scFv.

[0276] signal peptide In some embodiments of any aspect, the NK CAR polypeptide described herein comprises a signal peptide. A signal peptide may also be referred to as a signal, signal sequence, leader sequence, leader peptide, targeting signal, localization signal, localization sequence, or transit peptide. As used herein, the term "signal peptide" refers to the amino-terminal sequence of a polypeptide that directs a newly synthesized protein into the secretory pathway. Signal peptides typically consist of 13 to 36 somewhat hydrophobic amino acids. Signal peptides share a common structure: a short, positively charged amino-terminal region (n-region); a central hydrophobic region (h-region); and a highly polar carboxy-terminal region (c-region) that contains a site for cleavage by a signal peptidase. On the luminal side of the ER, the signal peptide is cleaved off by a signal peptidase. After successful folding of the nascent polypeptide by ER-resident chaperones and foldases, the protein is directed to exit the ER. This process may be supported by the presence of an N-terminal prosequence. While most type I membrane-bound proteins have a signal peptide, type II and multispanning membrane-bound proteins are targeted to the secretory pathway by a first transmembrane domain that is biochemically similar to a signal sequence, except that it is not cleaved.

[0277] In some embodiments of any aspect, the NK CAR polypeptides described herein comprise one, two, three, four, five, or more signal peptides. In some embodiments of any aspect, the CAR polypeptide or CAR system comprises one signal peptide, e.g., at the N-terminus of the polypeptide, prior to all other extracellular domains. In embodiments comprising multiple signal peptides, the multiple signal peptides can be different individual signal peptides, or multiple copies of the same signal peptide, or a combination thereof.

[0278] In some embodiments of any aspect, the signal peptide is a CD8a signal peptide. In some embodiments of any aspect, the signal peptide comprises SEQ ID NO:38 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:38 and maintains the same function as SEQ ID NO:38 (targeting of transmembrane proteins to the secretory pathway and cell membranes).

[0279] In some embodiments, the signal peptide of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising SEQ ID NO:37, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:37 and maintains the same function, or a nucleic acid sequence comprising a codon-optimized version of SEQ ID NO:37.

[0280] SEQ ID NOs:37 and 38 show the polynucleotide and amino acid sequences of exemplary CD8 signal peptides. TIFF2024504728000040.tif17159

[0281] Detectable Markers In some aspects, the present specification describes NK CAR polypeptides that contain at least one detectable marker. As used herein, the term "detectable marker" refers to a moiety that, when attached to a CAR polypeptide, confers detectability on the polypeptide or another molecule to which the polypeptide binds. In some embodiments of any aspect, the CAR polypeptide contains one, two, three, four, five, or more detectable markers. In some embodiments of any aspect, the CAR polypeptide or CAR system contains one detectable marker. In embodiments that contain multiple detectable markers, the multiple detectable markers can be different individual detectable markers, or multiple copies of the same detectable marker, or a combination thereof.

[0282] In some embodiments of any aspect, the detectable marker comprises an affinity tag.Non-limiting examples of affinity tags include Strep tag, chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), FLAG tag, HA tag, Myc tag, poly(His) tag, and their derivatives.In some embodiments of any aspect, the detectable marker is 3xFLAG (i.e., the FLAG motif, DYKDDDDK, SEQ ID NO: 131, is repeated three times).

[0283] In some embodiments of any aspect, a detectable marker of a CAR polypeptide described herein comprises one of SEQ ID NOs: 40, 122, 131, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs: 40, 122, or 131, and maintains the same function (e.g., detection of a CAR polypeptide) as one of SEQ ID NOs: 40, 122, or 131.

[0284] In some embodiments of any aspect, a detectable marker of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 39, 121, or a nucleic acid sequence comprising a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 39 or 121 and maintains the same function, or a nucleic acid sequence that comprises a codon-optimized version of one of SEQ ID NOs: 39 or 121.

[0285] SEQ ID NOs: 39, 40, 121 and 122 show the polynucleotide and amino acid sequences of exemplary FLAG tags (eg, 3xFlag). TIFF2024504728000041.tif36161

[0286] In some embodiments of any aspect, fluorescent moieties can be used as detectable markers, but detectable markers also include, for example, isotopes, fluorescent proteins and fluorescent peptides, enzymes, members of specific binding pairs, chromophores, affinity tags described herein or known in the art, antibodies, metal colloids (i.e., gold), and quantum dots. Detectable markers can be directly or indirectly detectable. Directly detectable markers do not require additional reagents or substrates to generate a detectable signal. Examples include isotopes and fluorophores. Indirectly detectable markers require the presence or action of one or more cofactors or substrates. Examples include enzymes such as β-galactosidase, which is detectable by the production of a colored reaction product upon cleavage of a substrate such as the chromogen X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside); horseradish peroxidase, which is detectable by the production of a colored reaction product in the presence of the substrate diaminobenzidine; and alkaline phosphatase, which is detectable by the production of a colored reaction product in the presence of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.

[0287] In some embodiments of any aspect, the detectable marker can be located anywhere within the CAR polypeptide described herein. In one embodiment, the detectable marker is located between any domain of the CAR polypeptide described herein, but is not found within the functional domain and does not interfere with the function of the domain. In some embodiments of any aspect, the detectable marker is located adjacent to and N-terminal to the extracellular binding domain. Such a marker can be used to detect expression of the CAR polypeptide, including cell surface expression. In some embodiments of any aspect, the detectable marker is located adjacent to one of the extracellular domains. In some embodiments of any aspect, the detectable marker is located adjacent to the transmembrane domain. In some embodiments of any aspect, the detectable marker is located adjacent to one of the intracellular domains.

[0288] In some embodiments of any aspect, the CAR polypeptides described herein, particularly those administered to a subject or that are part of a pharmaceutical composition, do not comprise a detectable marker that is immunogenic. In some embodiments of any aspect, the CAR polypeptides described herein do not comprise GFP, mCherry, HA1, or any other immunogenic marker.

[0289] Linker Domain In some aspects, the present specification describes NK CAR polypeptides that contain at least one linker domain. As used herein, a "linker domain" (used interchangeably with "peptide linker" or simply "linker") refers to an oligopeptide or polypeptide region, approximately 2 to 100 amino acids in length, that links together any of the domain sequences described herein. In some embodiments, the linker can contain or consist of flexible residues, such as glycine and serine, to allow adjacent protein domains to move freely relative to each other. Longer linkers can be used if it is desirable to ensure that two adjacent domains do not sterically interfere with each other. Linkers can be cleavable (e.g., designed to contain a substrate for a specific protease enzyme) or non-cleavable.

[0290] In some embodiments of any aspect, the NK CAR polypeptide described herein comprises one, two, three, four, five, or more linker domains. In some embodiments of any aspect, the NK CAR polypeptide comprises one linker domain. In embodiments comprising multiple linker domains, the multiple linker domains can be different individual linker domains, or multiple copies of the same linker domain, or a combination thereof. In some embodiments of any aspect, a linker peptide can be positioned between any two domains described herein, for example, between a detectable marker and a spacer domain, and / or between a VH / VL single-chain antigen-binding domain (e.g., in the case of an scFv). In some embodiments of any aspect, the linker domain is distal to the extracellular binding domain (i.e., further from the transmembrane domain than the extracellular binding domain). In some embodiments of any aspect, the linker domain is proximal to the signal peptide and / or detectable marker (i.e., closer to the transmembrane domain than the signal peptide and / or detectable marker).

[0291] A linker domain can comprise 1 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, 25 or more amino acids, 30 or more amino acids, 35 or more amino acids, 40 or more amino acids, 45 or more amino acids, 50 or more amino acids, or even more. Conversely, a linker domain can comprise fewer than 50 amino acids, fewer than 45 amino acids, fewer than 40 amino acids, fewer than 35 amino acids, fewer than 30 amino acids, fewer than 30 amino acids, fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, or fewer than 10 amino acids.

[0292] In some embodiments of the various aspects described herein, the linker domain comprises about 5 to about 50 amino acids. For example, the linker domain can comprise about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 amino acids, about 10 to about 30 amino acids, or about 15 to about 25 amino acids.

[0293] An exemplary linker domain is one consisting of glycine and serine residues, a so-called Gly-Ser polypeptide linker. As used herein, the term "Gly-Ser polypeptide linker" refers to a peptide consisting of glycine and serine residues. In some embodiments of the various aspects described herein, the linker domain has the amino acid sequence (Gly x Ser) n where x is 2, 3, 4, or 5 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, including, for example, SEQ ID NOs: 42, 120, 139-176 (see, e.g., Table 9).

[0294] Table 9: Exemplary linker sequences TIFF2024504728000042.tif63160

[0295] In some embodiments of any aspect, the linker domain of a CAR polypeptide described herein comprises one of SEQ ID NOs: 42, 120, 139-176, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 42, 120, 139-176 and maintains the same function (e.g., a flexible linker between domains of a CAR polypeptide) as one of SEQ ID NOs: 42, 120, 139-176.

[0296] In some embodiments of any aspect, a detectable marker of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 41, 119, or a nucleic acid sequence comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 41 or 119 and maintains the same function, or a nucleic acid sequence comprising a codon-optimized version of one of SEQ ID NOs: 41 or 119.

[0297] SEQ ID NOs:41, 42, 119 and 120 show the polynucleotide and amino acid sequences of exemplary linkers. TIFF2024504728000043.tif10157

[0298] spacer domain In some embodiments of any aspect, the NK CAR polypeptides described herein comprise a spacer domain. In some embodiments of any aspect, the spacer domain is located between the extracellular binding domain and the transmembrane domain. In some embodiments of any aspect, the spacer domain is C-terminal to the extracellular binding domain and N-terminal to the transmembrane domain. In some embodiments of any aspect, the spacer domain is N-terminal to the extracellular binding domain and C-terminal to the transmembrane domain. In some embodiments of any aspect, the spacer domain comprises a hinge domain. In some embodiments of any aspect, the CAR polypeptides described herein comprise one, two, three, four, five, or more hinge domains. In some embodiments of any aspect, the CAR polypeptides or CAR systems comprise one hinge domain. In embodiments comprising multiple hinge domains, the multiple hinge domains can be different individual hinge domains, multiple copies of the same hinge domain, or a combination thereof.

[0299] In some embodiments of any aspect, the hinge domain comprises a derivative of an immunoglobulin G (IgG)-based hinge or CD8α extracellular domain. Incorporation of a hinge domain has been shown to improve the expansion of chimeric antigen receptor T cells and increase the anti-tumor efficacy of CAR T cells (see, for example, Qin et al., Journal of Hematology & Oncology volume 10, Article number: 68 (2017); Stoiber et al., Cells. 2019 May; 8 (5): 472). The hinge domain can be derived from IgG subclasses (e.g., IgG1 and IgG4), IgD, and CD8 domains, of which IgG1 is the most widely used. The hinge domain preferably provides the following four aspects: (1) reduced binding affinity for Fcγ receptors, thereby reducing or eliminating off-target activation; (2) increased flexibility for the extracellular binding domain (e.g., scFv), thereby reducing spatial constraints between the tumor antigen and the CAR, and thus facilitating synapse formation between NK CAR cells and target cells; for example, to overcome steric hindrance in MUC1-specific CARs, a flexible and long hinge of an IgD isotype can be inserted; (3) reduced distance between the extracellular binding domain (e.g., scFv) and the target antigen or epitope; for example, anti-CD22 CARs require a hinge domain for optimal cytotoxicity; and (4) facilitates detection of CAR expression using anti-Fc reagents. In some embodiments of any aspect, the hinge domain promotes CAR dimerization.

[0300] In some embodiments of any aspect, the hinge domain comprises a CD8 hinge domain. In some embodiments of any aspect, the hinge domain comprises a CD8a hinge domain. In some embodiments of any aspect, the hinge domain of a CAR polypeptide described herein comprises SEQ ID NO:44 or an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:44, and maintains the same function as SEQ ID NO:44 (e.g., display of an extracellular binding region).

[0301] In some embodiments of any aspect, the hinge domain of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising SEQ ID NO:43, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:43 and maintains the same function, or a nucleic acid sequence comprising a codon-optimized version of SEQ ID NO:43.

[0302] SEQ ID NOs:43 and 44 show the polynucleotide and amino acid sequences of an exemplary CD8a hinge domain (e.g., residues 138-182 of CD8a; see, e.g., SEQ ID NO:138 for an exemplary full-length CD8a polypeptide sequence). TIFF2024504728000044.tif21160

[0303] Exemplary Polypeptides In aspects, described herein is an NK CAR polypeptide comprising any combination of domains described herein. In one aspect, described herein is a chimeric antigen receptor (CAR) polypeptide for expression in a natural killer (NK) cell comprising: (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one of: (i) an intracellular signaling domain derived from an NK cell receptor, (ii) an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein, and / or (iii) an intracellular costimulatory domain derived from a costimulatory receptor.

[0304] In one aspect, described herein is a chimeric antigen receptor (CAR) polypeptide (e.g., a Group I NK CAR polypeptide) for expression in a natural killer (NK) cell, comprising: (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one of (i) an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein and / or (ii) an intracellular costimulatory domain derived from a costimulatory receptor. In one aspect, described herein is a chimeric antigen receptor (CAR) polypeptide (e.g., a Group II-IV NK CAR polypeptide) for expression in a natural killer (NK) cell, comprising: (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one signaling domain derived from an NK cell receptor.

[0305] In some embodiments of any aspect, the polypeptide comprises, from N-terminus to C-terminus, (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain; such a polypeptide can also be referred to as a type I transmembrane protein. In some embodiments of any aspect, the CAR, which is a type I transmembrane protein, further comprises an N-terminal signal peptide. Non-limiting examples of such type I transmembrane protein CARs include CC002, CC003, CC004, CC005, CC007, CC008, CC013, CC016, CC017, CC018, CC024, CC026, CC027, CC029, CC030, CC032, CC033, CC034, CC035, CC037, CC038, CC039, CC103, CC104, CC108, CC118, CC124, CC130, CC135, which correspond to SEQ ID NOs: 80-90, 92-93, 95-96, 98-101, 103-110 and 112-113, respectively.

[0306] In some embodiments of any aspect, the polypeptide comprises, from C-terminus to N-terminus, (a) extracellular binding domain, (b) transmembrane domain, and (c) intracellular domain, and such polypeptide can also be referred to as type II transmembrane protein.In some embodiments of any aspect, the type II transmembrane protein CAR does not necessarily comprise N-terminal signal peptide.Non-limiting examples of such type II transmembrane protein CAR include CC025, CC028, CC031, CC036, CC125, and CC136, which correspond to SEQ ID NO:91, 94, 97, 102, 111, and 114, respectively.

[0307] In some embodiments of any aspect, the polypeptide is selected from the group consisting of Group Ic (e.g., CC005, CC007, CC008, or CC108), Group IIa (e.g., CC024, CC025, CC124, CC125), or Group IV (e.g., CC027, CC030, CC034, CC038, or CC130). In some embodiments of any aspect, the polypeptide is selected from the group consisting of Group Ic (e.g., CC005, CC007, CC008, or CC108), Group IIa (e.g., CC024, CC025, CC124, CC125), or Group IVd (e.g., CC030 or CC130). In some embodiments of any aspect, the polypeptide is selected from the group consisting of Group Ic (e.g., those containing cytokines, such as C007, CC008, or CC108), Group IIa (e.g., CC024, CC025, CC124, CC125), or Group IVd (e.g., CC030 or CC130).

[0308] In some embodiments of any aspect, the CAR polypeptide comprises one of SEQ ID NOs:80-114 (see, e.g., Table 6), or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of 80-114, while maintaining the same function (e.g., antigen binding and intracellular signaling). Table 6 provides exemplary CAR polypeptides and the specific order of their extracellular, transmembrane, and intracellular domains.

[0309] Table 6. Amino acid sequence numbers of constructs and domains ("TM" indicates transmembrane domain) TIFF2024504728000045.tif200159

[0310] Group I NK CAR polypeptide In one aspect, described herein is a Group I NK CAR polypeptide comprising: (a) an extracellular binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one of (i) an intracellular signaling domain derived from a NK cell membrane-bound signaling adaptor protein and / or (ii) an intracellular costimulatory domain derived from a costimulatory receptor. In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of CD8a.

[0311] In one aspect, described herein is a Group Ia NK CAR polypeptide comprising: (a) an extracellular binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular costimulatory domain derived from a costimulatory receptor. In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of CD8a. In some embodiments of any aspect, the costimulatory receptor is 4-1BB. In some embodiments of any aspect, the Group Ia NK CAR polypeptide is used as a control or reference.

[0312] In one aspect, the present specification describes a Group Ib NK CAR polypeptide comprising: (a) an extracellular binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising: (i) an intracellular signaling domain derived from a NK cell membrane-bound signaling adaptor protein; and (ii) an intracellular costimulatory domain derived from a costimulatory receptor. In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of CD8a. In some embodiments of any aspect, the costimulatory receptor is 4-1BB. In some embodiments of any aspect, the adaptor protein is CD3-zeta. In some embodiments of any aspect, the Group Ib NK CAR polypeptide is used as a control or a reference.

[0313] In one aspect, described herein is a Group Ic NK CAR polypeptide comprising: (a) an extracellular binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising: (i) an intracellular signaling domain derived from a NK cell membrane-bound signaling adaptor protein; and (ii) an intracellular costimulatory domain derived from a costimulatory receptor. In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of CD8a. In some embodiments of any aspect, the costimulatory receptor is 4-1BB. In some embodiments of any aspect, the adaptor protein is FcεRI. In some embodiments of any aspect, the Group Ic NK CAR polypeptide further comprises at least one cytokine (e.g., IL-15 and / or IL-21) and at least one self-cleaving peptide.

[0314] In one aspect, described herein is a Group Id NK CAR polypeptide comprising: (a) an extracellular binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising: (i) an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein; and (ii) two intracellular costimulatory domains derived from costimulatory receptors. In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of CD8a. In some embodiments of any aspect, the two costimulatory receptors are 4-1BB and IL2RB. In some embodiments of any aspect, the adaptor protein is CD3-zeta.

[0315] In one aspect, described herein is a Group Ie NK CAR polypeptide comprising: (a) an extracellular binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising: (i) an intracellular signaling domain derived from an NK cell membrane-bound signaling adaptor protein; and (ii) two intracellular costimulatory domains derived from costimulatory receptors. In some embodiments of any aspect, the transmembrane domain comprises the transmembrane domain of CD8a. In some embodiments of any aspect, the two costimulatory receptors are 4-1BB and IL2RB. In some embodiments of any aspect, the adaptor protein is FcεRI.

[0316] In some embodiments of any aspect, the Group I NK CAR polypeptide comprises one of SEQ ID NOs: 80-89 or 106-109, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the sequences of 80-89 or 106-109, and maintains the same function (e.g., antigen binding and intracellular signaling).

[0317] In some embodiments of any aspect, the Group I NK CAR polynucleotide comprises one of SEQ ID NOs:45-54 or 71-74, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:45-54 or 71-74 and maintains the same function as a polypeptide, or a codon-optimized version of one of SEQ ID NOs:45-54 or 71-74.

[0318] Anti-CD19 Group I Non-limiting examples of Group I NK CARs specific for CD19 include CC002, CC003, CC004, CC005, CC007, CC008, CC013, CC016, CC017, or CC018, which correspond to polynucleotide SEQ ID NOs:45-54 and polypeptide SEQ ID NOs:80-89, respectively. It should be understood that other NK CARs can be generated by replacing the CD-19 binding domain with a different tumor antigen binding domain or cell surface protein binding domain.

[0319] The CC002 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB. See, e.g., SEQ ID NO:45 or SEQ ID NO:80. TIFF2024504728000046.tif123161

[0320] The CC003 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB-CD3z. See, e.g., SEQ ID NO:46 or SEQ ID NO:81. TIFF2024504728000047.tif154161

[0321] The CC004 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB-CD3z. See, e.g., SEQ ID NO:47 or SEQ ID NO:82. TIFF2024504728000048.tif162161

[0322] The CC005 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB-FceR cytoplasmic domain. See, e.g., SEQ ID NO:48 or SEQ ID NO:83. TIFF2024504728000049.tif136161

[0323] The CC007 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB-FceR cytoplasmic domain-P2A peptide-IL15 isoform 1. See, e.g., SEQ ID NO:49 or SEQ ID NO:84. TIFF2024504728000050.tif188161

[0324] The CC008 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB-FceR cytoplasmic domain-P2A peptide-IL15 isoform 1-T2A-IL-21 isoform 1. See, e.g., SEQ ID NO:50 or SEQ ID NO:85. TIFF2024504728000051.tif177161TIFF2024504728000052.tif63161

[0325] The CC013 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB-IL2RB cytoplasmic domain-CD3z. See, e.g., SEQ ID NO:51 or SEQ ID NO:86. TIFF2024504728000053.tif184161

[0326] The CC016 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-CD8 transmembrane domain-IL2RB cytoplasmic domain-4-1BB-CD3z. See, e.g., SEQ ID NO:52 or SEQ ID NO:87. TIFF2024504728000054.tif184161

[0327] The CC017 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB-IL2RB cytoplasmic domain-FceR cytoplasmic domain. See, e.g., SEQ ID NO:53 or SEQ ID NO:88. TIFF2024504728000055.tif167161

[0328] The CC018 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-CD8 transmembrane domain-IL2RB cytoplasmic domain-4-1BB-FceR cytoplasmic domain. See, e.g., SEQ ID NO:54 or SEQ ID NO:89. TIFF2024504728000056.tif167161

[0329] Anti-CD33 Group I Non-limiting examples of Group I NK CARs specific for CD33 include CC103, CC104, CC108, or CC118, which correspond to polynucleotide SEQ ID NOs:71-74 and polypeptide SEQ ID NOs:106-109, respectively. It should be understood that other NK CARs can be generated by replacing the CD33 binding domain with a different tumor antigen binding domain or cell surface protein binding domain.

[0330] The CC103 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD33 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB-CD3z. See, e.g., SEQ ID NO:71 or SEQ ID NO:106. TIFF2024504728000057.tif151161

[0331] The CC104 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD33 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB-CD3z. See, e.g., SEQ ID NO:72 or SEQ ID NO:107. TIFF2024504728000058.tif160161

[0332] The CC108 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD33 scFv-CD8a hinge domain-CD8 transmembrane domain-4-1BB-FceR cytoplasmic domain-P2A peptide-IL15 isoform 1-T2A-IL-21 isoform 1. See, e.g., SEQ ID NO:73 or SEQ ID NO:108. TIFF2024504728000059.tif177161TIFF2024504728000060.tif60161

[0333] The CC118 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD33 scFv-CD8a hinge domain-CD8 transmembrane domain-IL2RB cytoplasmic domain-4-1BB-FceR cytoplasmic domain. See, e.g., SEQ ID NO:74 or SEQ ID NO:109. TIFF2024504728000061.tif164161

[0334] Group II-IV NK CAR polypeptides In one aspect, described herein are Group II-IV NK CAR polypeptides comprising (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one signaling domain derived from an NK cell receptor.

[0335] In one aspect, the present specification describes a Group II NK CAR polypeptide, which comprises (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one signaling domain derived from an NK cell receptor, wherein the transmembrane domain comprises the transmembrane domain of NKG2D. In some embodiments, the NK cell receptor comprises 2B4 (e.g., Group IIa). In some embodiments, the NK cell receptor comprises NTB-A (e.g., Group IIb). In some embodiments, the NK cell receptor comprises CD2 (e.g., Group IIc). In some embodiments, the NK cell receptor comprises CRACC (e.g., Group IId).

[0336] In one aspect, the present specification describes a Group III NK CAR polypeptide, which comprises (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one signaling domain derived from an NK cell receptor, wherein the transmembrane domain comprises the transmembrane domain of NKp46. In some embodiments, the NK CAR further comprises the intracellular signaling domain of NKp46. In some embodiments, the NK cell receptor comprises 2B4 (e.g., Group IIIa). In some embodiments, the NK cell receptor comprises NTB-A (e.g., Group IIIb). In some embodiments, the NK cell receptor comprises CD2 (e.g., Group IIIc). In some embodiments, the NK cell receptor comprises CRACC (e.g., Group IIId).

[0337] In one aspect, the present specification describes a Group IV NK CAR polypeptide comprising (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one signaling domain derived from an NK cell receptor, wherein the transmembrane domain comprises the transmembrane domain of DNAM1. In some embodiments, the NK CAR further comprises the intracellular signaling domain of DNAM1. In some embodiments, the NK cell receptor comprises 2B4 (e.g., Group IVa). In some embodiments, the NK cell receptor comprises NTB-A (e.g., Group IVb). In some embodiments, the NK cell receptor comprises CD2 (e.g., Group IVc). In some embodiments, the NK cell receptor comprises CRACC (e.g., Group IVd).

[0338] In some embodiments of any aspect, the Group II-IV NK CAR polypeptide comprises one of SEQ ID NOs: 90-105 or 110-114, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of 90-105 or 110-114, and maintains the same function (e.g., antigen binding and intracellular signaling).

[0339] In some embodiments of any aspect, the Group II-IV NK CAR polynucleotide comprises one of SEQ ID NOs:55-70 or 75-79, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:55-70 or 75-79 and has the same polypeptide function, or a codon-optimized version of one of SEQ ID NOs:55-70 or 75-79.

[0340] Anti-CD19 Group II-IV Non-limiting examples of Group II-IV NK CARs specific for CD19 include CC024, CC025, CC026, CC027, CC028, CC029, CC030, CC031, CC032, CC033, CC034, CC035, CC036, CC037, CC038, or CC039, which correspond to polynucleotide SEQ ID NOs:55-70 and polypeptide SEQ ID NOs:90-105, respectively. It should be understood that other NK CARs can be generated by replacing the CD19 binding domain with a different tumor antigen binding domain or cell surface protein binding domain.

[0341] The CC024 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-NKG2D transmembrane domain-2B4 cytoplasmic domain. See, e.g., SEQ ID NO:55 or SEQ ID NO:90. TIFF2024504728000062.tif149161

[0342] The CC025 construct (e.g., reverse domain order to CC024, except that it does not contain the CD8 signal peptide) comprises the following domains from N- to C-terminus: 2B4 cytoplasmic domain-NKG2D transmembrane domain-CD8a hinge domain-anti-CD19 scFv-linker (e.g., GGGGS, SEQ ID NO:42)-3*Flag. See, e.g., SEQ ID NO:56 or SEQ ID NO:91. TIFF2024504728000063.tif144161

[0343] The CC026 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-NKp46 transmembrane and cytoplasmic domain-CD2 cytoplasmic domain. See, e.g., SEQ ID NO:57 or SEQ ID NO:92. TIFF2024504728000064.tif152161

[0344] The CC027 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-transmembrane and cytoplasmic domains of DNAM1-2B4 cytoplasmic domain. See, e.g., SEQ ID NO:58 or SEQ ID NO:93. TIFF2024504728000065.tif166161

[0345] The CC028 construct (e.g., reverse domain order to CC039, except that it does not contain the CD8 signal peptide) comprises the following domains from N- to C-terminus: NTB-A cytoplasmic domain-NKG2D transmembrane domain-CD8a hinge domain-anti-CD19 scFv-linker (e.g., GGGGS, SEQ ID NO:42)-3*Flag. See, e.g., SEQ ID NO:59 or SEQ ID NO:94. TIFF2024504728000066.tif132161

[0346] The CC029 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-NKp46 transmembrane and cytoplasmic domains-NTB-A cytoplasmic domain. See, e.g., SEQ ID NO:60 or SEQ ID NO:95. TIFF2024504728000067.tif144161

[0347] The CC030 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-transmembrane and cytoplasmic domains of DNAM1-CRACC cytoplasmic domain. See, e.g., SEQ ID NO:61 or SEQ ID NO:96. TIFF2024504728000068.tif153161

[0348] The CC031 construct (e.g., reverse domain order to CC037, except that it does not contain the CD8 signal peptide) comprises the following domains from N- to C-terminus: CD2 cytoplasmic domain-NKG2D transmembrane domain-CD8a hinge domain-anti-CD19 scFv-linker (e.g., GGGGS, SEQ ID NO:42)-3*Flag. See, e.g., SEQ ID NO:62 or SEQ ID NO:97. TIFF2024504728000069.tif140161

[0349] The CC032 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-NKp46 transmembrane and cytoplasmic domain-2B4 cytoplasmic domain. See, e.g., SEQ ID NO:63 or SEQ ID NO:98. TIFF2024504728000070.tif154161

[0350] The CC033 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-NKp46 transmembrane and cytoplasmic domain-CRACC cytoplasmic domain. See, e.g., SEQ ID NO:64 or SEQ ID NO:99. TIFF2024504728000071.tif149161

[0351] The CC034 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-transmembrane and cytoplasmic domains of DNAM1-CD2 cytoplasmic domain. See, e.g., SEQ ID NO:65 or SEQ ID NO:100. TIFF2024504728000072.tif166161

[0352] The CC035 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-NKG2D transmembrane domain-CRACC cytoplasmic domain. See, e.g., SEQ ID NO:66 or SEQ ID NO:101. TIFF2024504728000073.tif136161

[0353] The CC036 construct (e.g., reverse domain order to CC035, except that it does not contain the CD8 signal peptide) comprises, from N- to C-terminus, the following domains: CRACC cytoplasmic domain-NKG2D transmembrane domain-CD8a hinge domain-anti-CD19 scFv-linker (e.g., GGGGS, SEQ ID NO:42)-3*Flag. See, e.g., SEQ ID NO:67 or SEQ ID NO:102. TIFF2024504728000074.tif131161

[0354] The CC037 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-NKG2D transmembrane domain-CD2 cytoplasmic domain. See, e.g., SEQ ID NO:68 or SEQ ID NO:103. TIFF2024504728000075.tif149161

[0355] The CC038 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-transmembrane and cytoplasmic domains of DNAM1-NTB-A cytoplasmic domain. See, e.g., SEQ ID NO:69 or SEQ ID NO:104. TIFF2024504728000076.tif153161

[0356] The CC039 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD19 scFv-CD8a hinge domain-NKG2D transmembrane domain-NTB-A cytoplasmic domain. See, e.g., SEQ ID NO:70 or SEQ ID NO:105. TIFF2024504728000077.tif136161

[0357] Anti-CD33 Group II-IV Non-limiting examples of Group II-IV NK CARs specific for CD33 include CC124, CC125, CC130, CC135, or CC136, which correspond to polynucleotide SEQ ID NOs:75-79 and polypeptide SEQ ID NOs:110-114, respectively. It should be understood that other NK CARs can be generated by replacing the CD33 binding domain with a different tumor antigen binding domain or cell surface protein binding domain.

[0358] The CC124 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD33 scFv-CD8a hinge domain-NKG2D transmembrane domain-2B4 cytoplasmic domain. See, e.g., SEQ ID NO:75 or SEQ ID NO:110. TIFF2024504728000078.tif147161

[0359] The CC125 construct (e.g., reverse domain order to CC124, except that it does not contain the CD8 signal peptide) comprises the following domains from N- to C-terminus: 2B4 cytoplasmic domain-NKG2D transmembrane domain-CD8a hinge domain-anti-CD33 scFv-linker (e.g., GGGGS, SEQ ID NO:42)-3*Flag. See, e.g., SEQ ID NO:76 or SEQ ID NO:111. TIFF2024504728000079.tif142161

[0360] The CC130 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD33 scFv-CD8a hinge domain-transmembrane and cytoplasmic domains of DNAM1-CRACC cytoplasmic domain. See, e.g., SEQ ID NO:77 or SEQ ID NO:112. TIFF2024504728000080.tif151161

[0361] The CC135 construct comprises the following domains from N- to C-terminus: CD8 signal peptide-3*Flag-linker (e.g., GGGGS, SEQ ID NO:42)-anti-CD33 scFv-CD8a hinge domain-NKG2D transmembrane domain-CRACC cytoplasmic domain. See, e.g., SEQ ID NO:78 or SEQ ID NO:113. TIFF2024504728000081.tif133161

[0362] The CC136 construct (e.g., reverse domain order to CC135, except that it does not contain the CD8 signal peptide) comprises the following domains from N- to C-terminus: CRACC cytoplasmic domain-NKG2D transmembrane domain-CD8a hinge domain-anti-CD33 scFv-linker (e.g., GGGGS, SEQ ID NO:42)-3*Flag. See, e.g., SEQ ID NO:79 or SEQ ID NO:114. TIFF2024504728000082.tif129161

[0363] Polynucleotides and Vectors In aspects, described herein are polynucleotides encoding NK CAR polypeptides. In some embodiments of any aspect, the CAR polynucleotide comprises one of SEQ ID NOs:45-79 (see, e.g., Table 5), or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:45-79, maintaining the same function as the polypeptide (e.g., antigen binding and intracellular signaling; see, e.g., SEQ ID NOs:80-114). Table 5 provides exemplary CAR polynucleotides and the specific order of their extracellular, transmembrane, and intracellular domains.

[0364] Table 5. Nucleic acid SEQ ID NOs for constructs and domains ("TM" indicates transmembrane domain) TIFF2024504728000083.tif200159

[0365] In some embodiments, the CAR polynucleotide is a codon-optimized version of SEQ ID NOs:145-79. In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized. For example, the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons, such that the altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but is transcribed and / or translated with improved efficiency in a desired expression system. In some embodiments of any aspect, the expression system is an organism (or cells derived from such an organism) other than the source of the native / wild-type sequence. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in mammals or mammalian cells, such as murine, murine, or human cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in human cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in yeast or yeast cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in bacterial cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in Escherichia coli (E. coli) cells.

[0366] In some embodiments, one or more of the genes described herein are expressed as a recombinant expression vector or plasmid. As used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring a transgene into a host cell. A vector can also include a viral particle carrying such a polynucleotide sequence. Vectors include, for example, plasmids, minichromosomes, phages, naked DNA, etc. See, for example, U.S. Patent Nos. 4,980,285, 5,631,150, 5,707,828, 5,759,828, 5,888,783, and 5,919,670, and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). One type of vector is a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, in which additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Furthermore, certain vectors are capable of directing the expression of genes operatively linked to the vector. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" are used interchangeably herein, as plasmids are the most commonly used form of vector. However, the technology described herein is intended to encompass other forms of expression vectors, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions and facilitate delivery of recombinant constructs, for example, to NK cells.

[0367] A cloning vector is one that can replicate in a host cell or become integrated into the genome in a host cell and further features one or more endonuclease endonuclease sites at which the vector can be cleaved in a determined manner and a desired DNA sequence ligated thereto so that the new recombinant vector retains its ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence can occur multiple times as the plasmid increases in copy number within a host cell, such as a host bacterium, or can occur only once per host until the host replicates by mitosis. In the case of a phage, replication can occur actively during a lytic phase or passively during a lysogenic phase.

[0368] An expression vector is one into which a desired DNA sequence can be inserted by restriction digestion and ligation so that the desired DNA sequence is operably linked to regulatory sequences and can be expressed as an RNA transcript. A vector can further contain one or more marker sequences suitable for use in identifying cells transformed or transfected with the vector or cells that have not been transformed or transfected. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activities can be detected by standard assays known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). In certain embodiments, the vectors used herein are capable of autonomous replication and expressing structural gene products present in the DNA segments to which they are operably linked.

[0369] As used herein, a coding sequence and a regulatory sequence are said to be "operably" linked when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequence be translated into a functional protein, two DNA sequences are said to be operably linked if the introduction of a promoter into the 5' regulatory sequences results in the transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region to direct transcription of the coding sequence, and (3) does not interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, a promoter sequence would be operably linked to a coding sequence if the promoter region is capable of effecting transcription of the DNA sequence in such a way that the resulting transcript can be translated into the desired protein or polypeptide.

[0370] When a nucleic acid molecule encoding any of the polypeptides described herein is expressed in a cell, a variety of transcription control sequences (e.g., promoter / enhancer sequences) can be used to direct its expression. The promoter can be a native promoter, i.e., a promoter endogenously associated with the gene, which provides normal regulation of expression of the gene. In some embodiments, the promoter can be constitutive, i.e., the promoter is unregulated and allows for continuous transcription of the gene associated with it. A variety of conditional promoters can also be used, e.g., promoters controlled by the presence or absence of a molecule.

[0371] The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but in general, they may include, as necessary, 5' non-transcribed and 5' non-translated sequences associated with initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed regulatory sequences include promoter regions, including promoter sequences for transcriptional control of operably linked genes. Regulatory sequences may also include enhancer sequences or upstream activating sequences, as desired. Vectors of the present invention may optionally include 5' leader or signal sequences. The selection and design of appropriate vectors is within the ability and discretion of one skilled in the art.

[0372] In some embodiments of any aspect, the promoter is a eukaryotic or human constitutive promoter, including, but not limited to, EF-1 alpha, SFFV, CMV, etc. In some embodiments of any aspect, the vector comprises the human elongation factor-1 alpha (EF-1 alpha) promoter, which is a constitutive promoter of human origin that can be used to drive ectopic gene expression under various in vitro and in vivo conditions. In some embodiments of any aspect, the vector comprises a silencing-prone spleen focus-forming virus (SFFV) promoter, which can result in higher levels of constitutive transgene expression compared to the CMV promoter or the EF1 alpha promoter. In some embodiments of any aspect, the vector comprises a Kozak sequence (e.g., GCCGCCACC), a nucleic acid motif that functions as a protein translation initiation site in eukaryotic mRNA transcripts.

[0373] Expression vectors containing all the elements necessary for expression are commercially available and known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by introducing heterologous DNA (RNA) into the cell. The heterologous DNA (RNA) is placed under the functional control of transcriptional elements so as to allow expression of the heterologous DNA in the host cell.

[0374] In some embodiments of any aspect, the vector is a lentiviral vector. The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and lentiviruses can deliver significant amounts of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery into mammalian cells and other cells. HIV, SIV, and FIV are all examples of lentiviruses. The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, inter alia, self-inactivating lentiviral vectors such as those described in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology and Lentigen's LENTIMAX™ vector system. Non-clinical types of lentiviral vectors are also available and would be known to those skilled in the art.

[0375] In some embodiments of any aspect, the lentiviral vector comprises a central polypurine tract (cPPT). The central polypurine tract / central termination sequence creates a "DNA flap" that increases nuclear import of the viral genome upon target cell infection. The cPPT / CTS element improves vector integration and transduction efficiency. In some embodiments of any aspect, the lentiviral vector comprises a marmot hepatitis virus posttranscriptional regulatory element (WPRE), which prevents poly(A) site readthrough, promotes RNA processing and maturation, and increases RNA nuclear export. In genomic transcripts, this enhances vector packaging and increases titer. In transduced target cells, the WPRE boosts transgene expression by facilitating mRNA transcript maturation.

[0376] In some embodiments, the vector contains a selectable marker, e.g., for selective amplification of the vector in bacteria. Non-limiting examples of selectable marker genes for use in bacteria include antibiotic resistance genes that confer resistance to ampicillin, tetracycline, and kanamycin. Tetracycline (tet) and ampicillin (amp) resistance marker genes can be obtained from any of several commercially available vectors, including pBR322 (available from New England BioLabs, Beverly, Massachusetts, catalog number 303-3s). The tet coding sequence is contained within nucleotides 86-476, and the amp gene is contained within nucleotides 3295-4155. The nucleotide sequence of the kanamycin (kan) gene is available from New England BioLabs' vector pACYC177, catalog number 401-L, GenBank accession number X06402.

[0377] In some embodiments, one or more of the recombinantly expressed genes may be integrated into the genome of the cell.

[0378] Nucleic acid molecules encoding the NK CAR polypeptides described herein can be introduced into one or more cells using standard methods and techniques in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation, including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing a nucleic acid molecule encoding an NK CAR polypeptide described herein can also be achieved by integrating the nucleic acid molecule into the genome.

[0379] cell In one aspect, described herein is a cell or population thereof comprising at least one NK CAR polypeptide described herein. In one aspect, described herein is a cell (e.g., NK cell) engineered to express a CAR, wherein the CAR NK cell exhibits anti-tumor properties. In one aspect, the cell is transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., NK cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express the CAR. In another embodiment, the cell (e.g., NK cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the CAR. In some such embodiments, the cell can transiently express the CAR.

[0380] In some embodiments of any aspect, the cells comprise immune cells. In some embodiments of any aspect, the immune cells comprise natural killer (NK) cells, CD4+ T cells, CD8+ T cells, or regulatory T cells (Tregs). In some embodiments of any aspect, the immune cells comprise natural killer (NK) cells.

[0381] In some embodiments of any aspect, the cells are isolated from a subject. As used herein, the term "isolated" refers to cells being placed in a state other than their native environment. The term "isolated" does not exclude the cells from being subsequently combined or mixed with other cells for later use. In some embodiments of any aspect, immune cells (e.g., NK cells) are (a) isolated from a subject, (b) genetically modified to express a CAR polypeptide described herein, and (c) administered to the subject. In some embodiments of any aspect, cells are isolated from a first subject and administered to a second subject. In some embodiments of any aspect, immune cells are first differentiated from a somatic cell sample from a subject and then genetically modified to express a CAR polypeptide described herein. In another embodiment, immune cells, e.g., NK cells, are differentiated from induced pluripotent stem cells derived from an individual, e.g., from an individual to whom the CAR-expressing NK cells described herein are to be administered.

[0382] In some embodiments, methods of genetically modifying cells to express a CAR can include, but are not limited to, the following methods: transfection or electroporation of cells with a vector encoding a CAR; transduction with a viral vector (e.g., retrovirus, lentivirus) encoding a CAR; gene editing using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganuclease-TALENs, or CRISPR-Cas; or any other method known in the art of genetically modifying cells to express a CAR.

[0383] Pharmaceutical Compositions and Administration In some embodiments, the methods described herein involve treating a subject who has or has been diagnosed with a disease or disorder (e.g., cancer, infectious disease, etc.) with an NK CAR polypeptide described herein. Subjects with such diseases or disorders can be identified by a physician using current diagnostic methods for cancer or infectious diseases. Symptoms and / or complications that characterize these conditions and aid in diagnosis are known in the art. A family history of cancer or infectious disease, or exposure to risk factors for cancer or infectious disease, can also determine whether a subject is likely to have such a disease or disorder, or aid in making a diagnosis of cancer or infectious disease.

[0384] The compositions described herein can be administered to a subject with or diagnosed with cancer or an infectious disease. In some embodiments, the methods described herein include administering to a subject an effective amount of a composition described herein, such as a CAR polypeptide described herein, to alleviate symptoms of cancer or an infectious disease. As used herein, "alleviating symptoms" refers to ameliorating any condition or symptom associated with cancer or an infectious disease. Compared to an equivalent untreated control, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, as measured by any standard technique.

[0385] Various means for administering the compositions described herein to a subject are known to those skilled in the art.The active substance can be administered intravenously by injection or by slow infusion over time.For example, given a suitable formulation for a given route, the active substance useful in the methods and compositions described herein can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, intratumorally, and if desired, can be delivered by peristaltic means or other means known to those skilled in the art.Local administration, for example, directly to the tumor site or lesion site, is also specifically contemplated.

[0386] The therapeutic composition that contains at least one active substance can be conventionally administered, for example, in unit dose.When the term "unit dose" is used in relation to therapeutic composition, it refers to a physically discrete unit that is suitable as a unit dosage for subject, and each unit contains a predetermined amount of active substance calculated to produce desired therapeutic effect, together with necessary physiologically acceptable diluent, i.e., carrier or vehicle.

[0387] Compositions are administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The amount and timing to be administered will depend on the subject being treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired.

[0388] The term "effective amount" as used herein refers to the amount of CAR-expressing NK cells described herein necessary to alleviate at least one or more symptoms of a disease or disorder, and relates to the amount of a pharmacological composition sufficient to provide the desired effect. Thus, the term "therapeutically effective amount" refers to the amount of CAR-expressing NK cells described herein sufficient to provide a specific anti-tumor effect (or anti-infectious disease effect) when administered to a typical subject. The effective amount used in various contexts herein may also include an amount sufficient to delay the onset of disease symptoms, alter the course of disease symptoms (for example, but not limited to, slow down the progression of disease symptoms), or reverse disease symptoms. Therefore, it is generally not feasible to specify an exact "effective amount." However, for any given case, one skilled in the art can determine an appropriate "effective amount" using only routine experimentation.

[0389] Effective doses, toxicity, and therapeutic efficacy can be determined by standard procedures in cell cultures or experimental animals, for example, to determine the minimum effective dose and / or the maximum tolerated dose. Dosages can vary depending on the dosage form used and the route of administration (e.g., systemic or intratumoral) utilized. A therapeutically effective dose can be initially estimated from cell culture assays. Dosages can also be formulated in animal models to achieve a dosage range between the minimum effective dose and the maximum tolerated dose. The effects of any particular dosage can be monitored by appropriate bioassays, such as assays for tumor growth and / or tumor size, among others. A physician can determine the dosage and adjust it, if necessary, to the observed effects of treatment.

[0390] Pharmaceutical compositions comprising the cells described herein, e.g., NK CAR cells, can be administered in a 10 2 ~10 10 cells / kg body weight, preferably 10 5 ~10 6 It can generally be said that the composition can be administered at a dosage of cells / kg body weight (including any integer value within these ranges). The number of cells will depend on the end use of the composition and the type of cells contained therein. For the uses provided herein, the cells are generally in a volume of 1 liter or less, which can be 500 mL or less, or even 250 mL or less, or 100 mL or less. Thus, the desired cell density is typically 10 6 cells / ml, typically >10 7 cells / ml, typically >10 8 cells / ml or more. A clinically relevant number of immune cells is cumulatively 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12In some aspects of the invention, the number of cells administered can be reduced to 10 or more, particularly since all of the infused cells will be redirected to a specific target antigen. 6 Pieces / kilogram (10 per patient) 6 ~10 11 In some embodiments, the dosage is about 1 x 10 per kg of body weight. 5 cells ~ approx. 1 x 10 8 In some embodiments, the dosage is about 1 x 10 cells per kg of body weight. 6 cells ~ approx. 1 x 10 7 In some embodiments, the dosage is about 1 x 10 cells per kg of body weight. 6 The CAR-expressing cell composition may be administered multiple times at dosages within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing treatment. If desired, the treatment may also include administration of a mitogen (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IL-10, IL-12, IL-15, IL-18, IL-21, IFN-γ, GM-CSF, TGF-β, TNF-α, and IFN-α, etc.) as described herein to enhance the induction of an immune response. In some embodiments, the administration of the cells may be performed once. In some embodiments, the administration of the cells may be repeated, for example, once, twice, or more times. In some embodiments, the administration of the cells may be performed daily, weekly, or monthly.

[0391] In some embodiments, the dose can be administered intravenously. In some embodiments, the intravenous administration can be an infusion administered over a period of about 10 minutes to about 3 hours. In some embodiments, the intravenous administration can be an infusion administered over a period of about 30 minutes to about 90 minutes.

[0392] In some embodiments, doses can be administered approximately once a week. In some embodiments, doses can be administered once a week. In some embodiments, doses can be administered once a week for about 12 weeks to about 18 weeks. In some embodiments, doses can be administered about every two weeks. In some embodiments, doses can be administered about every three weeks. In some embodiments, a total of about 2 to about 10 doses are administered. In some embodiments, a total of 4 doses are administered. In some embodiments, a total of 5 doses are administered. In some embodiments, a total of 6 doses are administered. In some embodiments, a total of 7 doses are administered. In some embodiments, a total of 8 doses are administered. In some embodiments, a total of 8 doses are administered. In some embodiments, the administration occurs over a total of about 4 weeks to about 12 weeks. In some embodiments, the administration occurs over a total of about 6 weeks. In some embodiments, the administration occurs over a total of about 8 weeks. In some embodiments, the administration occurs over a total of about 12 weeks. In some embodiments, the initial dose can be about 1.5 to about 2.5 times greater than subsequent doses.

[0393] Treatment according to the methods described herein can reduce the level of a marker or symptom of a condition, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0394] The doctor can determine the dosage of the compositions described herein and adjust it as necessary according to the observed effect of treatment.Regarding the duration and frequency of treatment, a skilled clinician will usually monitor the subject to determine when the treatment is providing therapeutic benefit, and decide whether to increase or decrease the dosage, increase or decrease the frequency of administration, interrupt treatment, resume treatment, or make other changes to the treatment regimen.

[0395] The dosage range for the CAR composition or NK CAR cell composition according to the methods described herein depends, for example, on the form of the CAR composition, its potency, and the degree to which symptoms, markers, or indicators of the conditions described herein are desired to be reduced, for example, the desired percentage reduction of the disease or disorder (e.g., cancer or infectious disease). The dosage should not be so high as to cause adverse side effects, such as autoimmunity. Generally, the dosage varies depending on the age, condition, and sex of the patient, and can be determined by one skilled in the art. If any complications occur, the dosage can also be adjusted by an individual physician.

[0396] The efficacy of a CAR composition, e.g., in treating a condition described herein, can be determined by a skilled clinician. However, a treatment is considered "effective" within the meaning of the present specification if, following treatment with the methods described herein, one or more of the signs or symptoms of a condition described herein are beneficially altered, other clinically acceptable symptoms are improved or even ameliorated, or a desired response is induced, e.g., by at least 10%. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or occurrence of the condition treated according to the methods described herein, or by measuring any other appropriate measurable parameter, e.g., tumor size. Efficacy can also be measured by the absence of deterioration of an individual (i.e., cessation of disease progression) as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those of skill in the art and / or are described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or animals), including (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation), or (2) reducing the severity of the disease, e.g., causing regression of symptoms. An effective amount for treating a disease means an amount sufficient to provide effective treatment for the disease as defined herein when administered to a subject in need thereof. The efficacy of an agent can be determined by assessing physical indicators of the condition or desired response (e.g., tumor size, pathogen titer, or antibody titer). Monitoring the efficacy of administration and / or treatment by measuring any one or any combination of such parameters is well within the capabilities of one of ordinary skill in the art. Efficacy can be assessed in animal models of the conditions described herein, e.g., animal models for cancer treatment. When using experimental animal models, efficacy of treatment is demonstrated when a statistically significant change is observed in a marker such as tumor size, pathogen titer, or antibody titer.

[0397] Provided herein are in vitro assays and animal model assays that allow for the evaluation of a given dose of CAR compositions. The efficacy of a given dosage combination can also be evaluated in an animal model, for example, in a specific cancer animal model.

[0398] In one aspect, described herein are pharmaceutical compositions comprising at least one CAR polypeptide, CAR polynucleotide, CAR vector, or CAR-containing cell described herein, collectively referred to as a "CAR composition." In some embodiments of any aspect, the pharmaceutical composition can include any combination of the CAR polypeptides described herein.

[0399] In some embodiments, the technology described herein relates to a pharmaceutical composition, comprising the CAR composition described herein and optionally a pharmaceutically acceptable carrier.In some embodiments, the active ingredient of the pharmaceutical composition comprises the CAR polypeptide described herein.Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer, solvent and / or dispersion medium.The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (24) C2-C 12Alcohols, such as ethanol; and (25) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "carrier," and "pharmaceutically acceptable carrier" are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent described herein, such as a CAR polypeptide.

[0400] In some embodiments, pharmaceutical compositions comprising the CAR compositions described herein can be in parenteral dosage forms.Since parenteral dosage forms typically bypass the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or can be sterilized before being administered to patients.Examples of parenteral dosage forms include, but are not limited to, ready-to-inject solutions, dry products that can be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, ready-to-inject suspensions, and emulsions.In addition, controlled-release parenteral dosage forms can be prepared for administration to patients, including, but not limited to, DUROS®-type dosage forms and dose-dumping dosage forms.

[0401] Suitable vehicles that can be used to provide parenteral dosage forms of the CAR compositions disclosed herein are well known to those skilled in the art. Examples include, but are not limited to, sterile water; USP Water for Injection; saline solution; glucose solution; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0402] Pharmaceutical compositions containing CAR compositions can also be formulated for oral administration, e.g., as discrete dosage forms, such as, but not limited to, tablets (including, but not limited to, scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, lozenges, wafers, aerosol sprays, or liquids, such as, but not limited to, syrups, elixirs, or solutions or suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions, or water-in-oil emulsions. Such compositions contain a predetermined amount of a pharmaceutically acceptable salt of a disclosed compound and can be prepared by pharmaceutical methods well known to those skilled in the art. For a general discussion, see Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia, PA (2005).

[0403] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, the use of conventional dosage forms may result in large fluctuations in drug concentrations in the patient's blood and other tissues. These fluctuations can affect several parameters, such as administration frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, and side effects. Advantageously, controlled-release formulations can be used to control the drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled-release or sustained-release dosage forms or formulations can be used to ensure that maximum drug efficacy is achieved while minimizing potential side effects and safety concerns that may arise from underdosing (i.e., below the minimum therapeutic level) or exceeding the drug's toxic levels. In some embodiments, the composition can be administered as a sustained-release formulation.

[0404] Controlled-release pharmaceutical products share a common goal: improving drug therapy compared to that achieved by their non-controlled counterparts. Ideally, the use of optimally designed controlled-release preparations in medical treatments is characterized by the use of a minimal amount of active pharmaceutical ingredient and the shortest possible time to cure or manage a condition. Advantages of controlled-release formulations include: 1) extended drug activity, 2) reduced dosing frequency, 3) improved patient compliance, 4) reduced total drug use, 5) reduced local or systemic side effects, 6) minimal drug accumulation, 7) reduced blood level fluctuations, 8) improved treatment efficacy, 9) reduced enhancement or loss of drug activity, and 10) improved speed of disease or condition management. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, PA, 2000).

[0405] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that immediately produces the desired therapeutic effect, and then gradually and continuously release another amount of drug so that that level of therapeutic or prophylactic effect is maintained over an extended period of time. To maintain this constant drug level in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions, including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0406] Various known controlled release or sustained release dosage forms, formulations and devices can be adapted for use with the salt and compositions of the present disclosure.Not limited to, examples include those described in United States Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, 5,733,566 and 6,365,185 B1, and these United States Patents are each incorporated herein by reference. These dosage forms can be used to provide sustained or controlled release of one or more active ingredients, for example, using hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (e.g., OROS® (Alza Corporation, Mountain View, Calif., USA)), or by combining them in various ratios to obtain the desired release profile.

[0407] In some embodiments of any aspect, the CAR compositions described herein are administered as a sole therapy, e.g., no other treatment for the disease or disorder (e.g., cancer) is administered to the subject.

[0408] In some embodiments of any aspect, the methods described herein can further include administering to the subject a second agent and / or treatment, e.g., as part of a combination therapy. Non-limiting examples of second agents and / or treatments include radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents, such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan. aziridines, such as benzodopa, carboquone, metuledopa, and uredopa; ethylenimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoamide, triethylenethiophosphoamide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adzelesin, carzelesin) , and synthetic analogs of bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, thiamin oxide, melphalan, novembicin, fenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enezyme antibiotics (e.g., calicheamicin, particularly calicheamicin gamma(l)I, calicheamicin theta(l)I, and / or calicheamicin omega(l)I (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; biphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norlo doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, isin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiampic acid Phosphorus, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole; elformithine; elliptinium acetate; epothilon; etoglucide; gallium nitrate; hydroxyurea; lentinan;Lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oregon; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazicone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, New Jersey), ABRAXANE® cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumburg, Illinois), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin);Examples of inhibitors include the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A, which reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0409] Those skilled in the art can easily identify useful chemotherapeutic agents (see, for example, Physicians' Cancer Chemotherapy Drug Manual 2014; Chapter 85 of Harrison's Principles of Internal Medicine, 18th Edition, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Chapters 28-29 of Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology in Principles of Cancer Therapy, Abeloff's Clinical Oncology, 2013, Elsevier; and Fischer DS (ed.): The Cancer Chemotherapy Handbook, 4th Edition, St. Louis, Mosby-Year Book, 2003).

[0410] Additionally, the method of treatment can further include the use of radiation or radiation therapy. Additionally, the method of treatment can further include the use of surgical procedures.

[0411] In some embodiments of any aspect, a CAR composition and an antimicrobial agent are administered to a subject. As used herein, the term "antimicrobial agent" (also referred to herein as antimicrobial drug, antimicrobial therapeutic drug, etc.) refers to a molecule or composition that destroys microorganisms (i.e., bacteria, fungi, viruses, parasites, and microbial spores) and prevents their development, growth, and / or pathogenic action. Thus, the term "antimicrobial agent" includes antibacterial drugs, antifungal drugs, and antiviral drugs. Exemplary antimicrobial agents include, but are not limited to, organic or inorganic small molecules; peptides; proteins; peptide analogs and peptide derivatives; peptidomimetics; antibodies (polyclonal or monoclonal); antigen-binding fragments of antibodies; nucleic acids; nucleic acid analogs and nucleic acid derivatives; extracts made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; natural or synthetic compositions; and any combination thereof.

[0412] In some embodiments of any aspect, the antimicrobial agent can be selected from aminoglycosides, ansamycins, beta-lactams, bis-biguanides, carbacephems, carbapenems, cationic polypeptides, cephalosporins, fluoroquinolones, glycopeptides, iron-sequestering glycoproteins, linosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quaternary ammonium compounds, quinolones, silver compounds, sulfonamides, tetracyclines, and any combination thereof. In some embodiments of any aspect, the antimicrobial agent can include an antibiotic.

[0413] In some embodiments of any aspect, the subject is administered a CAR composition and an antibacterial agent. Specific examples of antibacterial agents include broad-spectrum penicillins, amoxicillins (e.g., ampicillin, bacampicillin, carbenicillin indanyl, mezlocillin, piperacillin, ticarcillin), penicillins, and beta-lactamase inhibitors (e.g., amoxicillin-clavulanic acid, ampicillin-sulbactam, benzylpenicillin, cloxacillin, dicloxacillin, methicillin, oxacillin, penicillin G, penicillin, methicillin, oxacillin, penicillin, penicillin G, penicillin ... nicillin V, piperacillin-tazobactam, ticarcillin-clavulanic acid, nafcillin), cephalosporins (e.g., first-generation cephalosporins, cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, cephradine), second-generation cephalosporins (e.g., cefaclor, cefamandole, cefonicid, cefotetan, cefoxitin, cefprozil, cefmetazole, cefroxil, cephalosporins of third generation (e.g., cefdinir, ceftibuten, cefoperazone, cefixime, cefotaxime, cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone), cephalosporins of fourth generation (e.g., cefepime), macrolides and lincosamides (e.g., azithromycin, clarithromycin, clindamycin, dirithromycin, quinolones and fluoroquinolones (e.g., cinoxacin, ciprofloxacin, enoxacin, gatifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, oxolinic acid,acid), gemifloxacin, perfloxacin), carbapenems (e.g., imipenem-cilastatin, meropenem), monobactams (e.g., aztreonam), aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin), glycopeptides (e.g., teicoplanin, vancomycin), tetracyclines (e.g., demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline, chlortetracycline), sulfonamides (e.g., mafenide, silver sulfadiazine, sulfacetamide, sulfadiazine, sulfamethoxazole, These include toxazole, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfamethizole), rifampins (e.g., rifabutin, rifampin, rifapentine), oxazolidinones (e.g., linezolid, streptogramins, quinupristin, dalfopristin), bacitracin, chloramphenicol, fosfomycin, isoniazid, methenamine, metronidazole, mupirocin, nitrofurantoin, nitrofurazone, novobiocin, polymyxin, spectinomycin, trimethoprim, colistin, cycloserine, capreomycin, ethionamide, pyrazinamide, para-aminosalicylic acid, and erythromycin ethylsuccinate.

[0414] In some embodiments of any aspect, the subject is administered a CAR composition and an antiviral agent. In some embodiments of any aspect, the antiviral agent is abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir, antiretroviral, arbidol, atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, dolutegravir, ecoliever, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, favipiravir, fomivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitor, ganciclovir, hydroxychloroquine, ibacitabine, idoxuridine, imiquimod, imunovir, indinavir, inosine, integrase inhibitor, interferon, interferon type I, interferon type II, interferon type III, lamivudine, ram ... thiazolidine, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide, novir, nucleoside analogues, oseltamivir (Tamiflu), peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, pyramidine, raltegravir, remdesivir, and thiazolidine. The antiviral agent may be selected from the group consisting of transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, sofosbuvir, stavudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, Truvada, valacyclovir (Valtrex), valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza) and zidovudine.See, for example, PCT patent applications WO2019018185A1, WO2019079339A1, WO2020055368A2, US patent application publication US20200017514A1, and US patent US8575195.The contents of each of these documents are incorporated herein by reference in their entirety.Other antiviral agents known in the art may also be used.

[0415] Treatment method The NK CAR compositions described herein can be administered to a subject in need thereof, particularly in the treatment of cancer. In some embodiments of any aspect, the CAR compositions described herein can be administered to treat cancer or infectious diseases. Infectious diseases that can be treated with the CAR compositions described herein include any microorganism that has a specific microbial antigen that can be targeted, and the infectious disease can be a viral infection or a bacterial infection.

[0416] In some embodiments, the method of treatment can include first diagnosing a subject or patient who can benefit from treatment with a composition described herein. In some embodiments, such diagnosis includes detecting or measuring an abnormal level of a marker (e.g., a tumor antigen or an antigen from a virus or bacterium described herein) in a sample from the subject or patient. In some embodiments, the method further includes administering a CAR composition described herein to the patient.

[0417] In some embodiments, the level of the analyte described herein is determined to be abnormal in comparison with the standard in advance.In some embodiments, the standard level can be the level in the sample obtained from the sample with similar cell type, sample type, sample processing, and / or the sample obtained from the subject with similar age, sex and other demographic parameters compared with the sample / subject.In some embodiments, the test sample and the control reference sample are of the same cell type, i.e., obtained from the same biological source, and comprise the same composition, for example, comprise the same number and the same type of cell.

[0418] As used herein, the term "sample" or "test sample" refers to a sample taken or isolated from an organism, such as a blood or plasma sample from a subject. In some embodiments of any aspect, the technology described herein encompasses several examples of biological samples. In some embodiments of any aspect, the biological sample is a cell, or tissue, or peripheral blood, or bodily fluid. Exemplary biological samples include, but are not limited to, biopsies, tumor samples, biofluid samples; blood; serum; plasma; urine; sperm; mucus; tissue biopsies; organ biopsies; synovial fluid; bile; cerebrospinal fluid; mucosal secretions; exudates; sweat; saliva; and / or tissue samples. This term also includes mixtures of the above-mentioned samples. The term "test sample" also includes untreated or pretreated (or preprocessed) biological samples. In some embodiments of any aspect, the test sample can include cells from a subject.

[0419] In some embodiments of any aspect, determining whether a subject has an abnormal level of an analyte described herein can include i) obtaining a sample from the subject, or having a sample already obtained from the subject, and ii) performing an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject, or having such an assay already performed. In some embodiments of any aspect, determining whether a subject has an abnormal level of an analyte described herein can include performing an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject, or having such an assay already performed. In some embodiments of any aspect, determining whether a subject has an abnormal level of an analyte described herein includes ordering or requesting an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any aspect, determining whether a subject has an abnormal level of an analyte described herein includes receiving the results of an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any aspect, determining whether a subject has an abnormal level of an analyte described herein can include receiving a report, result, or other means identifying the subject as having a decreased level of the analyte.

[0420] In one aspect, described herein is a method of treating a subject in need of a CAR-based therapy, the method comprising administering to the subject a therapeutically effective amount of a CAR-based therapy selected from the group consisting of an NK CAR polypeptide described herein, an NK CAR polynucleotide described herein, an NK CAR vector described herein, an NK CAR lentivirus described herein, an NK CAR cell or population thereof described herein, or an NK CAR pharmaceutical composition described herein.

[0421] In one aspect of any of these embodiments, described herein is a method of treating cancer or an infectious disease in a subject in need thereof, comprising: a) determining whether the subject has an abnormal level of an analyte (e.g., a tumor antigen, a viral antigen, or a bacterial antigen described herein); and b) if the level of the analyte is increased or otherwise abnormal compared to a reference standard, instructing or instructing the subject that a CAR composition described herein should be administered. In some embodiments of any aspect, instructing or instructing the subject that a particular treatment should be administered can include providing a report of the assay results. In some embodiments of any aspect, instructing or instructing the subject that a particular treatment should be administered can include providing a report of the assay results and / or a treatment recommendation that takes the assay results into account. In some embodiments of any aspect, administration of the CAR-based therapy results in increased specific lysis of cancer cells targeted by the CAR. In some embodiments of any aspect, administration of the CAR-based therapy results in increased specific lysis of infected cells targeted by the CAR.

[0422] In one aspect, described herein is a method of increasing activation of an NK cell or population thereof, comprising contacting the cell or population thereof with an NK CAR polypeptide described herein, an NK CAR polynucleotide described herein, an NK CAR vector described herein, or an NK CAR lentivirus described herein.

[0423] In some embodiments of any aspect, contacting NK cells or a population thereof with the polypeptide, polynucleotide, vector, or lentivirus increases the activity of the NK cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to before contact with the polypeptide, polynucleotide, vector, or lentivirus. In some embodiments of any aspect, increased activation of NK cells or a population thereof comprises increased expression of a cytokine or granzyme selected from the group consisting of TNFα, IFNγ, GM-CSF, and granzyme B.

[0424] In some embodiments of any aspect, increased activation of NK cells or a population thereof results in increased specific lysis of target cells. In some embodiments of any aspect, the target cells express a surface antigen that specifically binds to the extracellular binding domain of the polypeptide. In some embodiments of any aspect, the target cells are cancer cells. In some embodiments of any aspect, the target cells are cells infected with a virus or bacteria.

[0425] In another aspect, described herein is a method of producing a therapeutic composition, the method comprising introducing an NK CAR polypeptide described herein, an NK CAR polynucleotide described herein, an NK CAR vector described herein, or an NK CAR lentivirus described herein into an NK cell under conditions that allow expression of said NK CAR polypeptide in the NK cell.

[0426] In some embodiments of any aspect, the NK cells are removed from the subject in need of the therapeutic composition prior to introduction of the nucleic acid, polynucleotide, vector, or lentivirus. In some embodiments of any aspect, the NK cells are returned to the subject after introduction of the nucleic acid, polynucleotide, vector, or lentivirus.

[0427] cancer In some embodiments of the various aspects described herein, the CAR polypeptides described herein can be used to treat cancer. As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and invade nearby tissues. Cancer cells can spread to other parts of the body via the blood and lymphatic systems. There are several major types of cancer. Carcinoma is cancer that occurs in the skin or tissues lining or covering internal organs. Sarcoma is cancer that occurs in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemia is cancer that occurs in hematopoietic tissues, such as bone marrow, and causes abnormal blood cells to be produced in large quantities and enter the blood. Lymphoma and multiple myeloma are cancers that occur in cells of the immune system. Central nervous system cancer is cancer that occurs in the tissues of the brain and spinal cord.

[0428] In some embodiments of any aspect, the cancer is a primary cancer. In some embodiments of any aspect, the cancer is a malignant cancer. As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibit one or more of the following: uncontrolled growth (i.e., dividing beyond normal limits), invasion (i.e., invading and destroying adjacent tissues), and metastasis (i.e., spreading to other parts of the body via the lymph or blood). As used herein, the term "metastasizing" refers to the spread of cancer from one part of the body to another. Tumors formed by spread cells are called "metastatic tumors" or "metastases." Metastatic tumors contain the same cells as in the original (primary) tumor. As used herein, the terms "benign" or "non-malignant" refer to tumors that can grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[0429] "Cancer cell" or "tumor cell" refers to an individual cell of a cancerous tumor or cancerous tissue. A tumor generally refers to a swelling or lesion formed by the abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, such as leukemia, do not necessarily form tumors. In the case of tumor-forming cancer cells, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0430] As used herein, the term "neoplasm" refers to any new, abnormal growth of tissue, such as an abnormal mass of tissue whose growth is excessive and uncoordinated with that of normal tissue. Thus, a neoplasm can be a benign, premalignant, or malignant neoplasm.

[0431] A subject with cancer or tumor is a subject in whose body there are objectively measurable cancer cells. This definition includes malignant, actively growing cancers as well as potentially dormant tumors or micrometastases. Cancer that migrates from its original location and disseminates to other vital organs may ultimately lead to the death of the subject due to the functional deterioration of the affected organs.

[0432] Examples of cancer include carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma (GBM); liver cancer; hepatoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer Cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; and other carcinomas and sarcomas; and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nevus syndrome, edema (such as that associated with brain tumors), and Meigs syndrome.

[0433] In some embodiments of any aspect, the subject has or has been diagnosed with adrenal gland cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, gallbladder cancer, gestational trophoblastic disease, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, sarcoma, soft tissue sarcoma, spinal cancer, stomach cancer, testicular cancer, pharyngeal cancer, tumor, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0434] "Cancer cells" are cancerous, precancerous, or transformed cells in vivo, ex vivo, or in tissue culture that have spontaneous or induced phenotypic changes that do not necessarily require the incorporation of new genetic material. Transformation can occur through infection with a transforming virus and incorporation of new genomic nucleic acid, or through the incorporation of exogenous nucleic acid, but can also occur spontaneously or after exposure to carcinogens that mutate endogenous genes. Transformation / cancer is associated with cellular morphological changes, immortalization, dysregulation of growth, foci formation, anchorage independence, malignant lesions, loss of contact inhibition and density restriction of growth, growth factor or serum independence, tumor-specific markers, invasion or metastasis, and tumor growth in appropriate animal hosts, such as nude mice.

[0435] infectious disease In some embodiments of the various aspects described herein, the CAR polypeptides described herein can be used to treat infectious diseases. In some embodiments of the various aspects described herein, the infectious disease is a bacterial infection. In some embodiments of the various aspects described herein, the infectious disease is a viral infection.

[0436] Non-limiting examples of pathogenic bacteria include spirochetes (e.g., Borrelia), actinomycetes (e.g., Actinomyces), mycoplasmas, rickettsiae, gram-negative aerobic bacilli, gram-negative aerobic cocci, gram-negative facultative anaerobic bacilli (e.g., Erwinia and Yersinia), gram-negative cocci, gram-negative cocci, gram-positive cocci (e.g., Staphylococcus and Streptococcus), endospore-forming bacilli, and endospore-forming cocci. Further non-limiting examples of bacterial pathogens include species of the genera Bacillus, Brucella, Burkholderia, Francisella, Yersinia, Streptococcus, Haemophilus, Neisseria, Listeria, Clostridium, Klebsiella, Legionella, Escherichia (e.g., E. coli), Mycobacterium, Staphylococcus, Campylobacter, Vibrio, and Salmonella, as well as drug-resistant and multi-drug-resistant and highly virulent strains of these pathogenic bacteria. Non-limiting examples of known foodborne bacterial pathogens include members of the genera Salmonella, Clostridium, Campylobacter, Staphylococcus, Salmonella, Escherichia (e.g., E. coli), and Listeria.In some embodiments, non-limiting examples of bacterial pathogens include Bacillus anthracis, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, Streptococcus groups A and B, MRSA, Streptococcus pneumonia, Haemophilus influenza, Neisseria meningitides, Listeria monocytogenes, Clostridium difficile, Klebsiella, highly virulent pathogenic strains of Escherichia coli, Mycobacterium tuberculosis, and the like. tuberculosis, Staphylococcus aureus, Campylobacter spp., Salmonella spp., and Clostridium perfringens, as well as drug-resistant, multi-drug-resistant, and hypervirulent strains of these pathogenic bacteria. In some embodiments, non-limiting examples of known foodborne bacterial pathogens include Salmonella, non-typhoidal Clostridium perfringens, Campylobacter spp., Staphylococcus aureus, Salmonella, non-typhoidal Campylobacter spp., Escherichia coli (STEC) 0157, and Listeria monocytogenes.

[0437] In some embodiments of any aspect, the CAR polypeptides described herein are used to treat RNA viruses. By way of non-limiting example, the RNA viruses are group III (dsRNA) viruses, group IV (+ssRNA) viruses, group V (-ssRNA) viruses, or group VI (reverse transcriptase) viruses.

[0438] In some embodiments of any aspect, the RNA virus is a Group III (i.e., double-stranded RNA (dsRNA)) virus. In some embodiments of any aspect, the Group III RNA virus belongs to a virus family selected from the group consisting of Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae (e.g., rotavirus), Totiviridae, and Quadriviridae. In some embodiments of any aspect, the Group III RNA virus belongs to the genus Botybirnavirus. In some embodiments of any aspect, the Group III RNA virus is an unassigned species selected from the group consisting of Botrytis porri RNA virus 1, Circulifer tenellus virus 1, Colletotrichum camelliae filamentous virus 1, Cucurbit yellows associated virus, Sclerotinia sclerotiorum decline associated virus, and Spissistilus festinus virus 1.

[0439] In some embodiments of any aspect, the RNA virus is a Group IV (i.e., positive-sense single-stranded (ssRNA)) virus. In some embodiments of any aspect, the Group IV RNA virus belongs to a viral order selected from the group consisting of Nidovirales, Picornavirales, and Tymovirales. In some embodiments of any aspect, the Group IV RNA viruses are classified into the following families: Arteriviridae, Coronaviridae (e.g., coronaviruses, SARS-CoV), Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae (e.g., poliovirus, rhinovirus (common cold virus), hepatitis A virus), Secoviridae (e.g., Comovirinae), Alphaflexiviridae, Betaflexiviridae, and Gammaflexiviridae. Gammaflexiviridae, Tymoviridae, Alphatetraviridae, Alvernaviridae, Astroviridae, Barnaviridae, Benyviridae, Bromoviridae, Caliciviridae (e.g., Norwalk virus), Carmotetraviridae, Closteroviridae, Flaviviridae (e.g., yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, Zika virus), Fusariviridae,The virus belongs to a viral family selected from the group consisting of Hepeviridae, Hypoviridae, Leviviridae, Luteoviridae (e.g., Barley Yellow Dwarf Virus), Polycipiviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Potyviridae, Sarthroviridae, Statovirus, Togaviridae (e.g., Rubella Virus, Ross River Virus, Sindbis Virus, Chikungunya Virus), Tombusviridae, and Virgaviridae. In some embodiments of any aspect, the Group IV RNA virus belongs to a viral genus selected from the group consisting of Bacillariornavirus, Dicipivirus, Labyrnavirus, Sequiviridae, Blunervirus, Cilevirus, Higrevirus, Idaeovirus, Negevirus, Ourmiavirus, Polemovirus, Sinaivirus, and Sobemovirus. In some embodiments of any aspect, the Group IV RNA virus is selected from the group consisting of Acyrthosiphon pisum virus, Bastrovirus, Blackford virus, blueberry necrotic ring blotch virus, Cadicistrovirus, Chara australis virus,Extra small virus, Goji berry yellows virus, Hepelivirus, Jingmen tick virus, Le Blanc virus, Nedicistrovirus, Nesidiocoris tenuis virus 1, Niflavirus, Nylanderia fulva virus 1, Orsay virus, Osedax japonicus RNA virus 1, Picalivirus, Plasmopara halstedii virus, Rosellinia necatrix fusarivirus 1, Santeuil virus, Secalivirus, Solenopsis invicta virus 3, Wuhan large pig roundworm In some embodiments of any aspect, the Group IV RNA virus is a satellite virus selected from the group consisting of the Sarusulaviridae family, the genus Albetovirus, the genus Aumaivirus, the genus Papanivirus, the genus Virtovirus, and chronic bee paralysis virus.

[0440] In some embodiments of any aspect, the RNA virus is a Group V (i.e., negative-sense ssRNA) virus. In some embodiments of any aspect, the Group V RNA virus belongs to a phylum or subphylum selected from the group consisting of Negarnaviricota, Haploviricotina, and Polyploviricotina. In some embodiments of any aspect, the Group V RNA virus belongs to a class of viruses selected from the group consisting of Chunqiuviricetes, Ellioviricetes, Insthoviricetes, Milneviricetes, Monjiviricetes, and Yunchangviricetes. In some embodiments of any aspect, the Group V RNA virus belongs to a viral order selected from the group consisting of Articulavirales, Bunyavirales, Goujianvirales, Jingchuvirales, Mononegavirales, Muvirales, and Serpentovirales.In some embodiments of any aspect, the Group V RNA virus is selected from the group consisting of Amnoonviridae (e.g., Taastrup virus), Arenaviridae (e.g., Lassa virus), Aspiviridae, Bornaviridae (e.g., Borna disease virus), Chuviridae, Cruliviridae, Feraviridae, Filoviridae (e.g., Ebola virus, Marburg virus), Fimoviridae, Hantaviridae, Jonviridae, Mymonaviridae, Nairoviridae, Nyamiviridae, and the like. ridae, Orthomyxoviridae (e.g., influenza virus), Paramyxoviridae (e.g., measles virus, mumps virus, Nipah virus, Hendra virus, and NDV), Peribunyaviridae, Phasmaviridae, Phenuiviridae, Pneumoviridae (e.g., RSV and metapneumovirus), Qinviridae, Rhabdoviridae (e.g., rabies virus), Sunviridae, Tospoviridae, and Yueviridae.In some embodiments of any aspect, the Group V RNA virus belongs to a viral genus selected from the group consisting of Anphevirus, Arlivirus, Chengtivirus, Crustavirus, Tilapineviridae, Wastrivirus, and Deltavirus (e.g., Hepatitis D virus).

[0441] In some embodiments of any aspect, the RNA virus is a Group VI RNA virus that contains a virally encoded reverse transcriptase. In some embodiments of any aspect, the Group VI RNA virus belongs to the order Ortervirales. In some embodiments of any aspect, the Group VI RNA virus belongs to a virus family or virus subfamily selected from the group consisting of Belpaoviridae, Caulimoviridae, Metaviridae, Pseudoviridae, Retroviridae (e.g., a retrovirus, e.g., HIV), Orthoretrovirinae, and Spumaretrovirinae.In some embodiments of any aspect, the Group VI RNA virus is an Alpharetrovirus (e.g., avian leukosis virus; Rous sarcoma virus), a Betaretrovirus (e.g., mouse mammary tumor virus), a Bovispumavirus (e.g., bovine foamy virus), a Deltaretrovirus (e.g., bovine leukemia virus; human T-lymphotropic virus), an Epsilonretrovirus (e.g., walleye dermal sarcoma virus), an Equispumavirus (e.g., equine foamy virus), a Felispumavirus (e.g., feline foamy virus), a Gammaretrovirus (e.g., murine leukemia virus, feline leukemia virus), a Lentivirus (e.g., human immunodeficiency virus 1, simian immunodeficiency virus, feline immunodeficiency virus), a Prosimiispumavirus (e.g., Brown greater galago prosimian leukemia ... In some embodiments of any aspect, the virus belongs to a viral genus selected from the group consisting of endogenous retrovirus (ERV; e.g., endogenous retrovirus group W envelope member 1 (ERVWE1); HCP5 (HLA complex P5); human teratocarcinoma-derived virus), which are endogenous viral elements in the genome that are closely related and can be derived from retroviruses.

[0442] In some embodiments of any aspect, the CAR polypeptides described herein are used to treat DNA viruses. By way of non-limiting example, the DNA viruses are group I (dsDNA) viruses, group II (ssDNA) viruses, or group VII (dsDNA-RT) viruses.

[0443] In some embodiments of any aspect, the DNA virus is a Group I (i.e., dsDNA) virus. In some embodiments of any aspect, the Group I dsDNA virus belongs to a viral order selected from the group consisting of Caudovirales, Herpesvirales, and Ligamenvirales. In some embodiments of any aspect, the Group I dsDNA viruses include viruses from the Adenoviridae (e.g., adenovirus), Alloherpesviridae, Ampullaviridae, Ascoviridae, Asfarviridae (e.g., African swine fever virus), Baculoviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, and Herpesviridae (e.g., human herpesvirus, varicella-zoster virus, and varicella-zoster virus). Herpes viruses), Hytrosaviridae, Iridoviridae, Lavidaviridae, Lipothrixviridae, Malacoherpesviridae, Marseilleviridae, Mimiviridae, Myoviridae (e.g., enterobacteriaceae phage T4), Nimaviridae, Nudiviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae,The virus belongs to a virus family selected from the group consisting of Podoviridae (e.g., enterobacterial phage T7), Polydnavirus, Polyomaviridae (e.g., Simian virus 40, JC virus, BK virus), Poxviridae (e.g., cowpox virus, variola), Rudiviridae, Siphoviridae (e.g., enterobacterial phage λ), Sphaerolipoviridae, Tectiviridae, Tristromaviridae, and Turriviridae. In some embodiments of any aspect, the Group I dsDNA virus belongs to a virus genus selected from the group consisting of Dinodnavirus, Rhizidiovirus, and Salterprovirus. In some embodiments of any aspect, the Group I dsDNA virus belongs to an unassigned viral species selected from the group consisting of Abalone shriveling syndrome-associated virus, Apis mellifera filamentous virus, Bandicoot papillomatosis carcinomatosis virus, Cedrat virus, Cowmoeva virus, KIs-V, lentil virus, Leptopilina boulardi filamentous virus, Megavirus, Metallosphaera turreted icosahedral virus, Methanosarcina spherical virus, Mollivirus sibericum virus, Orpheovirus IHUMI-LCC2, Phaeocystis globosa virus, and Pithovirus. In some embodiments of any aspect, the Group I dsDNA virus is Organic Lake virophage,The virophage is selected from the group consisting of Ace Lake Mavirus virophage, Dishui Lake virophage 1, Guarani virophage, Phaeocystis globosa virus virophage, Rio Negro virophage, Sputnik virophage 2, Yellowstone Lake virophage 1, Yellowstone Lake virophage 2, Yellowstone Lake virophage 3, Yellowstone Lake virophage 4, Yellowstone Lake virophage 5, Yellowstone Lake virophage 6, Yellowstone Lake virophage 7, and Zamilon virophage 2.

[0444] In some embodiments of any aspect, the DNA virus is a Group II (i.e., ssDNA) virus. In some embodiments of any aspect, the Group II ssDNA virus belongs to a virus family selected from the group consisting of Anelloviridae, Bacilladnaviridae, Bidnaviridae, Circoviridae, Geminiviridae, Genomoviridae, Inoviridae, Microviridae, Nanoviridae, Parvoviridae, Smacoviridae, and Spiraviridae.

[0445] In some embodiments of any aspect, the DNA virus is a Group VII (i.e., dsDNA-RT) virus. In some embodiments of any aspect, the Group VII dsDNA-RT virus belongs to the order Ortervirales. In some embodiments of any aspect, the Group VII dsDNA-RT virus belongs to the family Caulimoviridae or Hepadnaviridae (e.g., hepatitis B virus). In some embodiments of any aspect, the Group VII dsDNA-RT virus belongs to a viral genus selected from the group consisting of Badnavirus, Caulimovirus, Cavemovirus, Petuvirus, Rosadnavirus, Solendovirus, Soymovirus, Tungrovirus, Avihepadnavirus, and Orthohepadnavirus.

[0446] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are set forth below. Unless otherwise stated or necessitated by context, the following terms and phrases include the meanings set forth below. These definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed invention, as the scope of the present invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition set forth herein, the definition set forth herein shall prevail.

[0447] For convenience, certain terms employed in the specification, examples, and appended claims of this application are collected here.

[0448] The terms "reduce," "reduce," "reduction," or "inhibit" are all used herein to refer to a statistically significant decrease. In some embodiments, "reduce," "reduction," "reduce," or "inhibit" typically refers to a decrease of at least 10% compared to a baseline level (e.g., in the absence of a given treatment or agent), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or complete reduction compared to a baseline level. "Complete inhibition" is 100% inhibition compared to a baseline level. The decrease can preferably be to a level that is accepted as within the normal range, for example, for individuals without a given disorder.

[0449] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a base level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any increase between 10-100% compared to a base level, or at least about 2-fold, at least about 3-fold, at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase compared to a base level, or any increase between 2-fold and 10-fold or more. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0450] As used herein, the term "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, felines, such as domestic cats, canines, such as dogs, foxes, wolves, birds, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, for example, a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0451] Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects corresponding to animal models of cancer or infectious diseases. The subject can be male or female.

[0452] The subject may be a subject who has been previously diagnosed or identified as suffering from or having a condition (e.g., cancer or an infectious disease) requiring treatment or one or more complications related to such a condition, and optionally has already received treatment for the disease or disorder (e.g., cancer or an infectious disease) or one or more complications related to the disease or disorder (e.g., cancer or an infectious disease). Alternatively, the subject may not have been previously diagnosed with the disease or disorder (e.g., cancer or an infectious disease) or one or more complications related to the disease or disorder (e.g., cancer or an infectious disease). For example, the subject may exhibit one or more risk factors for the disease or disorder (e.g., cancer or an infectious disease) or one or more complications related to the disease or disorder (e.g., cancer or an infectious disease), or may not exhibit risk factors.

[0453] A "subject in need" of treatment for a particular condition can be a subject who has the condition, a subject who has been diagnosed with the condition, or a subject who is at increased risk of developing the condition.

[0454] As used herein, the terms "protein" and "polypeptide" are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, although the usage of these terms overlaps in the art. The terms "protein" and "polypeptide" are used interchangeably herein to refer to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs thereof.

[0455] The various aspects described herein are further intended to encompass variants (whether naturally occurring or not), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any of the specific polypeptides described. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions in a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small number of amino acids in the encoded sequence are "conservatively modified variants" if the changes result in the substitution of amino acids with chemically similar amino acids and retain the desired activity of the polypeptide. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0456] Specific polypeptide domains, regions, or amino acid residues that are likely to be tolerant of change can be identified by aligning different intra- or inter-species variants of the polypeptide (e.g., an NK cell receptor, an NK cell membrane-bound signaling adaptor protein, or a costimulatory receptor described herein) or domains thereof (e.g., an intracellular signaling domain, a costimulatory domain, a transmembrane domain, etc.). Domains, regions, or amino acid residues that exhibit little intra- and / or inter-species variation represent conserved sequences, which are likely to be unable to tolerate substantial change or mutation or to tolerate only conservative substitutions. Domains, regions, or amino acid residues that exhibit intra- and / or inter-species variation represent non-conserved sequences, which are likely to be able to tolerate change or mutation (e.g., non-conservative or conservative substitutions).

[0457] For example, a given amino acid can be replaced with a residue having similar physicochemical characteristics, such as substituting one aliphatic residue for another (e.g., Ile, Val, Leu, or Ala for another), or one polar residue for another (e.g., Lys for Arg, Glu for Asp, or Gln for Asn). Other such conservative substitutions are known, such as substituting entire regions with similar hydrophobic characteristics. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, e.g., the function and specificity of the native or reference polypeptide, is retained.

[0458] Amino acids can be grouped according to the similarity of the properties of their side chains (Allehninger, Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped based on shared side chain characteristics: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would entail exchanging a member of one of these classes for another. Specific conservative substitutions include, for example, Ala to Gly or Ser, Arg to Lys, Asn to Gln or His, Asp to Glu, Cys to Ser, Gln to Asn, Glu to Asp, Gly to Ala or Pro, His to Asn or Gln, Ile to Leu or Val, Leu to Ile or Val, Lys to Arg, to Gln or Glu, Met to Leu, Tyr or Ile, Phe to Met, Leu or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp, and / or Phe to Val, Ile, or Leu.

[0459] In some embodiments, a polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide in an assay known in the art or described herein. A functional fragment can include one or more conservative substitutions of a sequence disclosed herein. In some embodiments of any aspect, the polypeptide can include an initial N-terminal amino acid, methionine. In embodiments where a polypeptide does not include an initial N-terminal methionine, it is understood that a variant of the polypeptide includes an initial N-terminal methionine.

[0460] In some embodiments, the polypeptides described herein can be variants of the sequences described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutation of the native nucleotide sequence. As used herein, a "variant" is a polypeptide that is substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from the native or reference polypeptide due to one or more deletions, insertions, or substitutions. DNA sequences encoding variant polypeptides include sequences that contain one or more nucleotide additions, deletions, or substitutions compared to the native or reference DNA sequence, but encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0461] The amino acid or DNA sequence of a variant can be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to the native or reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined by comparing the two sequences using computer programs commonly available for this purpose on the World Wide Web, freely available (e.g., BLASTp or BLASTn, with default settings).

[0462] The amino acid sequence of a variant can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more similar to a native or reference sequence. As used herein, "similarity" refers to identical amino acids or amino acids conservatively substituted as described herein. Thus, a percentage of "sequence similarity" refers to the percentage of identical or conservatively changed amino acids, e.g., "sequence similarity" = (% sequence identity) + (% conservative changes). It should be understood that a sequence having a specified percent similarity to a reference sequence necessarily encompasses a sequence having the same specified percent identity to the reference sequence. Those skilled in the art will be aware of several different computer programs that use different mathematical algorithms and can be used to determine the identity or similarity between two sequences. For example, computer programs using the Needleman and Wunsch algorithm (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego, USA); the algorithm of E. Meyers and W. Miller (Meyers et al. (1989)) incorporated into the ALIGN program (version 2.0); or, more preferably, BLAST (Basic Local Alignment Tool, using default parameters) can be utilized. See, e.g., U.S. Patent No. 10,023,890, the contents of which are incorporated herein by reference in their entirety.

[0463] Modification of the native amino acid sequence can be achieved by any of several techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing mutant sequences flanked by restriction sites that allow ligation to fragments of the native sequence. The resulting reconstructed sequence after ligation encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis can be used to obtain modified nucleotide sequences with specific codons modified according to the required substitution, deletion, or insertion. Techniques for making such modifications are well established and include those disclosed, for example, in Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entireties. Any cysteine ​​residue not involved in maintaining the proper conformation of the polypeptide can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) can be added to a polypeptide to improve its stability or facilitate oligomerization.

[0464] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, that incorporates ribonucleic acid, deoxyribonucleic acid, or analogue units. Nucleic acid can be single-stranded or double-stranded. Single-stranded nucleic acid can be one nucleic acid strand of denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, nucleic acid can be DNA. In another aspect, nucleic acid can be RNA. Suitable DNA can include, for example, genomic DNA, cDNA, or vector DNA. Suitable RNA can include, for example, mRNA.

[0465] The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and, as appropriate, protein secretion, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as appropriate. Expression can refer to the transcription and stable accumulation of sense RNA (e.g., mRNA) or antisense RNA derived from one or more nucleic acid fragments, and / or the translation of mRNA into polypeptides.

[0466] As used herein, the term "detect" or "measure" refers to observing a signal, for example, from a probe, a label, or a target molecule, to indicate the presence of an analyte in a sample. Any method known in the art for detecting a specific label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopy, fluorescence, photochemistry, biochemistry, immunochemistry, electrical, optical, or chemical methods. In some embodiments of any aspect, measurement can be a quantitative observation.

[0467] In some embodiments of any aspect, the polypeptides, nucleic acids, or cells described herein can be engineered. As used herein, "engineered" refers to the aspect of being manipulated by human hands. For example, a polypeptide is considered "engineered" if at least one aspect of the polypeptide, such as its sequence, has been manipulated by human hands to be different from the aspect of its natural state. As is customary in the art and understood by those skilled in the art, the progeny of an engineered cell can still be referred to as "engineered," even if the actual manipulation was performed on a previous entity.

[0468] In some embodiments of any aspect, the CAR polypeptide described herein is exogenous. In some embodiments of any aspect, the CAR polypeptide described herein is ectopic. In some embodiments of any aspect, the CAR polypeptide described herein is not endogenous.

[0469] The term "exogenous" refers to a substance present in a cell that is not its original source. As used herein, the term "exogenous" can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide introduced into a biological system, such as a cell or organism, by a process involving human intervention, where the nucleic acid or polypeptide is not normally found in the biological system and a human desires to introduce the nucleic acid or polypeptide into the cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide introduced into a biological system, such as a cell or organism, by a process involving human intervention, where the nucleic acid or polypeptide is normally found in relatively low amounts in the biological system and a human desires to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is natural to a biological system or cell. As used herein, "ectopic" refers to a substance found in an abnormal location and / or amount. An ectopic substance can also be one that is normally found in a given cell, but in a much lower amount and / or at a different time. Ectopic also includes substances such as polypeptides or nucleic acids that are not naturally found or expressed in a given cell in its natural environment.

[0470] As used herein, an "antigen" is a molecule bound by a binding site on an antibody agent (e.g., an extracellular binding domain containing an antibody-derived structure). Typically, an antigen is bound by an antibody ligand and has the ability to generate an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid, or other molecule, or a localized portion thereof. The term "antigenic determinant" refers to the epitope on an antigen that is recognized by an antigen-binding molecule, more specifically, by the antigen-binding site of the molecule.

[0471] In some embodiments, the nucleic acid encoding a polypeptide described herein (e.g., a CAR polypeptide) is contained in a vector. In some aspects described herein, the nucleic acid sequence encoding a given polypeptide described herein or any module thereof is operably linked to a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. As used herein, a vector can be a viral vector or a non-viral vector. The term "vector" encompasses any genetic element that, when associated with the proper control elements, is capable of replication and can transfer a gene sequence into a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, etc.

[0472] In some embodiments of any aspect, the vector is a recombinant vector. For example, the vector contains sequences originating from at least two different sources or has been otherwise modified by human hands. In some embodiments of any aspect, the vector contains sequences originating from at least two different species. In some embodiments of any aspect, the vector contains sequences originating from at least two different genes. For example, the vector contains a nucleic acid encoding a fusion protein or a gene product operably linked to at least one non-native (e.g., heterologous) gene control element (e.g., promoter, suppressor, activator, enhancer, response element, etc.).

[0473] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The sequences to be expressed are often, but not necessarily, heterologous to the cell. Expression vectors may contain additional elements. For example, an expression vector may have two replication systems, allowing it to be maintained in two organisms, such as human cells for expression and prokaryotic hosts for cloning and amplification.

[0474] The term "viral vector" as used herein refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. A viral vector can contain a nucleic acid encoding a polypeptide described herein instead of a non-essential viral gene. The vector and / or particle can be used to transfer any nucleic acid into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Non-limiting examples of viral vectors useful in the technology disclosed herein include AAV vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

[0475] It should be understood that in some embodiments, the vector described herein can be combined with other suitable compositions and treatments.In some embodiments, the vector is episomal.The use of suitable episomal vectors is a way to maintain the nucleotide of interest in the extrachromosomal DNA of high copy number in the subject, thereby eliminating the potential effect of chromosomal integration.

[0476] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatment aimed at reversing, alleviating, ameliorating, inhibiting, slowing, or halting the progression or severity of a condition associated with a disease or disorder, e.g., cancer or an infectious disease. The term "treating" encompasses reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder (e.g., cancer or an infectious disease). Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" encompasses not only the improvement of symptoms or markers, but also the halting or at least slowing of the progression or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, diminishment of the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, remission or palliation of the disease state (whether partial or complete), and / or reduced mortality. The term "treatment" of a disease also encompasses the alleviation of symptoms or side effects of the disease (including palliative treatment).

[0477] As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier, such as a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to a compound, material, composition and / or dosage form that is suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response or other problems or complications, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. In some embodiments of any aspect, the pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any aspect, the pharmaceutically acceptable carrier can be an artificial carrier or an engineered carrier, for example, a carrier in which the active ingredient is not found in nature.

[0478] As used herein, the term "administering" refers to introducing a compound disclosed herein into a subject by a method or route that results in at least partial delivery of the active substance at the desired site. Pharmaceutical compositions, including compositions disclosed herein, can be administered by any suitable route that results in effective treatment in the subject. In some embodiments, administration involves physical human activity, such as injection, application, and / or operation of a delivery device or delivery apparatus. Such activity can be performed, for example, by a medical professional and / or the subject being treated.

[0479] As used herein, "contacting" refers to any suitable means for delivering or exposing an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery into cell culture medium, transfection, transduction, perfusion, injection, or other delivery methods known to those skilled in the art. In some embodiments, the contacting step includes physical human activity, such as injection, dispensing, mixing, and / or decanting, and / or operating a delivery device or delivery apparatus.

[0480] The terms "statistically significant" or "significant" refer to statistical significance, generally meaning a difference of 2 standard deviations (2 SD) or greater.

[0481] Except in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in each instance by the term "about." When used in conjunction with percentages, the term "about" can mean ±1%.

[0482] As used herein, the term "comprising" means that in addition to the specified elements present, other elements may also be present. The use of "comprising" indicates inclusion rather than limitation.

[0483] The term "consisting of" refers to the compositions, methods, and individual components thereof described herein, excluding any element not recited in the description of that embodiment.

[0484] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0485] As used herein, the term "corresponding to" refers to the amino acid or nucleotide at a numbered position in a first polypeptide or nucleic acid being equivalent to the amino acid or nucleotide at the numbered position in a second polypeptide or nucleic acid. Equivalent numbered amino acids or nucleotides can be determined by alignment of candidate sequences using homology programs known in the art, such as BLAST.

[0486] As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells, and / or particles, in which a first entity binds to a second target entity with higher specificity and affinity than when it binds to a third, non-target entity. In some embodiments, specific binding can refer to the affinity of a first entity for a second target entity that is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times, or more than its affinity for a third, non-target entity. A reagent specific for a given target exhibits specific binding to that target under the conditions of the assay used.

[0487] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia," and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with "for example."

[0488] Groupings of alternative elements or aspects of the invention disclosed herein should not be construed as limitations. The members of each group may be referred to and claimed individually or in any combination with other members of that group or other elements found herein. One or more members of a group may be included in or excluded from a group for reasons of convenience and / or patentability. When any such inclusion or exclusion is made, the specification is deemed to include the group as modified and thus satisfy the written description of all Markush groups used in the claims.

[0489] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that the present invention is not limited to the particular methods, protocols, and reagents described herein, which may, as such, vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims of this application. Definitions of common terms in immunology and molecular biology can be found in "The Merck Manual of Diagnosis and Therapy," 20th Edition, Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); "The Encyclopedia of Molecular Cell Biology and Molecular Medicine," Robert S. Porter et al. (eds.), Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and "Molecular Biology and Biotechnology: a Comprehensive Desk Reference," Robert A. Meyers (ed.), VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); "Immunology," Werner Luttmann, Elsevier, 2006; "Janeway's Immunobiology," Kenneth Murphy, Allan Mowat, Casey Weaver (ed.), W.W.Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); "Lewin's Genes XI" Jones & Bartlett Publishers, 2014 (ISBN-1449659055); "Molecular Cloning: A Laboratory Manual" 4th ed. by Michael Richard Green and Joseph Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); "Basic Methods in Molecular Biology" by Davis et al., Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); "Laboratory Methods in Enzymology: DNA" edited by Jon Lorsch, Elsevier, 2013 (ISBN 0124199542); "Current Protocols in "Current Protocols in Protein Science" (CPPS), edited by John E. Coligan, John Wiley and Sons, Inc., 2005; and "Current Protocols in Immunology" (CPI), edited by John E. Coligan, A.D. M. Kruisbeek, David H. Margulies, Ethan M. Shevach, and Warren Strobe, John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entireties.

[0490] In some embodiments of any aspect, the disclosure described herein does not relate to processes for cloning humans, processes for modifying the genetic identity of human germ lines, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that are likely to cause suffering to the animals without providing any substantial medical benefit to humans or animals, nor to the animals resulting from such processes.

[0491] Other terms are defined herein within the description of various aspects of the invention.

[0492] All patents and other publications referred to in this application, including literature references, issued patents, published patent applications, and co-pending patent applications, are specifically incorporated herein by reference to describe and disclose, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for the purpose of their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior invention or for any other reason. Any statement as to the date of these documents or representation as to the contents of these documents is based on information available to the applicants and does not constitute an admission as to the correctness of the dates or contents of these documents.

[0493] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments or examples of the present disclosure have been described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as will be apparent to those skilled in the relevant art. For example, while method steps or functions may be presented in a given order, alternative embodiments may perform the functions in a different order, or functions may be performed substantially simultaneously. The teachings of the disclosure provided herein may be applied to other procedures or methods, as appropriate. The various embodiments described herein may be combined to provide additional embodiments. If necessary, aspects of the present disclosure may be modified to utilize the compositions, functions, and concepts of the above-mentioned references and applications to provide additional embodiments of the present disclosure. Furthermore, considerations of biological functional equivalence allow for certain changes to be made to protein structures without affecting the type or amount of biological or chemical activity. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0494] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Furthermore, although advantages associated with certain embodiments of the present disclosure have been described in connection with those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages, to be encompassed within the scope of the present disclosure.

[0495] Certain aspects of the technology described herein can be defined according to the following numbered paragraphs. 1. a) the extracellular binding domain; b) a transmembrane domain, and c) i) an intracellular signaling domain derived from the NK cell receptor; ii) an intracellular signaling domain derived from a NK cell membrane-associated signaling adaptor protein, and / or iii) Intracellular costimulatory domains derived from costimulatory receptors an intracellular domain comprising at least one of A chimeric antigen receptor (CAR) polypeptide for expression in a natural killer (NK) cell, comprising: 2. NK cell receptors a) i) Natural killer cell receptor 2B4, ii) Natural killer cell-T cell-B cell antigen (NTB-A), iii) cytotoxic cell-activating CD2-like receptor (CRACC), and iv) cluster of differentiation antigens 2 (CD2), or b) the intracellular signaling domain comprises the amino acid sequence of one of SEQ ID NOs: 7-10 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 7-10; 10. The polypeptide of paragraph 1. 3. NK cell membrane-bound signaling adaptor protein a) i) high affinity IgE receptor (FcεR1) and ii) CD3-zeta (CD3ζ) or b) the intracellular signaling domain comprises the amino acid sequence of one of SEQ ID NOs: 11-12 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 11-12; 10. The polypeptide of paragraph 1. 4. Costimulatory receptors a) 4-1BB and / or IL2 receptor beta (IL2RB), or b) the intracellular costimulatory domain comprises an amino acid sequence of one of SEQ ID NOs: 15-16 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 15-16; 10. The polypeptide of paragraph 1. 5. 10. The polypeptide of paragraph 1, wherein the intracellular domain further comprises at least one self-cleaving peptide and / or cytokine. 6. The self-cleaving peptide a) T2A, P2A, E2A, or F2A; or b) comprising the amino acid sequence of one of SEQ ID NOs: 19-20 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 19-20; 6. The polypeptide of paragraph 5. 7. The cytokines a) IL-15 or IL-21; b) comprises the amino acid sequence of one of SEQ ID NOs: 23-24 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 23-24; or c) adjacent to and distal to the self-cleaving peptide such that the cytokine is separated from the polypeptide by the self-cleaving peptide; 6. The polypeptide of paragraph 5. 8. The transmembrane domain is a) the transmembrane domain of the native NK cell receptor; b) the transmembrane domain of CD8, or c) an amino acid sequence of one of SEQ ID NOs: 29 to 32 or 117 to 118, or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 29 to 32 or 117 to 118 2. The polypeptide of paragraph 1, comprising: 9. 9. The polypeptide of paragraph 8, wherein the transmembrane domain of a native NK cell receptor is selected from the group consisting of natural killer group 2D (NKG2D), natural killer cell P46-related protein (NKp46), and DNAX accessory molecule-1 (DNAM1). 10. The extracellular binding domain is a) an antibody, an antigen-binding fragment thereof, a F(ab) fragment, a F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb); b) specifically binds to a tumor-associated antigen, or c) comprising an amino acid sequence of one of SEQ ID NOs: 35 to 36 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 35 to 36; 10. The polypeptide of paragraph 1. 11. 2. The polypeptide of paragraph 1, further comprising a signal peptide located N-terminal to the extracellular binding domain. 12. The signal peptide is a) the CD8 signal peptide, or b) comprising the amino acid sequence of SEQ ID NO:38 or an amino acid sequence that is at least 80% identical to SEQ ID NO:38; 12. The polypeptide of paragraph 11. 13. 10. The polypeptide of paragraph 1, further comprising a detectable marker distal to the extracellular binding domain. 14. 12. The polypeptide of paragraph 11, further comprising a linker domain distal to the extracellular binding domain and / or proximal to the signal peptide. 15. 10. The polypeptide of paragraph 1, further comprising a spacer domain positioned between the extracellular binding domain and the transmembrane domain. 16. The spacer domain is a) the CD8 hinge domain, or b) the amino acid sequence of SEQ ID NO:44 or an amino acid sequence that is at leas...

Claims

1. a) the extracellular binding domain; b) a transmembrane domain, and c) i) an intracellular signaling domain derived from the NK cell receptor; ii) an intracellular signaling domain derived from a NK cell membrane-associated signaling adaptor protein, and / or iii) Intracellular costimulatory domains derived from costimulatory receptors an intracellular domain comprising at least one of A chimeric antigen receptor (CAR) polypeptide for expression in a natural killer (NK) cell, comprising:

2. NK cell receptors a) i) Natural killer cell receptor 2B4, ii) Natural killer cell, T cell, and B cell antigen (NTB-A); iii) cytotoxic cell-activating CD2-like receptor (CRACC), and iv) cluster of differentiation antigen 2 (CD2), or b) the intracellular signaling domain comprises an amino acid sequence of one of SEQ ID NOs:7-10 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:7-10; The polypeptide of claim 1.

3. NK cell membrane-associated signaling adaptor protein, a) i) High affinity IgE receptor (FcεR1) and ii) CD3-zeta (CD3ζ) or b) the intracellular signaling domain comprises an amino acid sequence of one of SEQ ID NOs:11-12 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:11-12; The polypeptide of claim 1.

4. Costimulatory receptors a) 4-1BB and / or IL2 receptor beta (IL2RB), or b) the intracellular costimulatory domain comprises an amino acid sequence of one of SEQ ID NOs: 15-16 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 15-16; The polypeptide of claim 1.

5. The polypeptide of any one of claims 1 to 4, wherein the intracellular domain further comprises at least one self-cleaving peptide and / or cytokine.

6. The self-cleaving peptide is a) T2A, P2A, E2A, or F2A; or b) comprising an amino acid sequence of one of SEQ ID NOs: 19-20 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 19-20; The polypeptide of claim 5.

7. The cytokines a) IL-15 or IL-21; b) comprises the amino acid sequence of one of SEQ ID NOs:23-24 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs:23-24; or c) adjacent to and distal to the autocleaving peptide such that the cytokine is separated from the polypeptide by the autocleaving peptide; The polypeptide of claim 5.

8. The transmembrane domain is a) a transmembrane domain of a native NK cell receptor selected from the group consisting of natural killer group 2D (NKG2D), natural killer cell P46-related protein (NKp46), and DNAX accessory molecule-1 (DNAM1); b) the transmembrane domain of CD8, or c) an amino acid sequence of one of SEQ ID NOs: 29-32 or 117-118 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 29-32 or 117-118 The polypeptide according to any one of claims 1 to 7, comprising:

9. The extracellular binding domain is a) an antibody, an antigen-binding fragment thereof, F(ab) fragment, F(ab') fragment, single chain variable fragment (scFv), or single domain antibody (sdAb); b) specifically binds to a tumor-associated antigen, or c) comprising an amino acid sequence of one of SEQ ID NOs: 35-36 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 35-36; A polypeptide described in any one of claims 1 to 8.

10. The polypeptide according to any one of claims 1 to 9, further comprising a signal peptide located on the N-terminal side of the extracellular binding domain, a detectable marker distal to the extracellular binding domain, a linker domain distal to the extracellular binding domain and / or proximal to the signal peptide, and / or a spacer domain located between the extracellular binding domain and the transmembrane domain.

11. The signal peptide is a) the CD8 signal peptide, or b) comprising the amino acid sequence of SEQ ID NO:38 or an amino acid sequence that is at least 80% identical to SEQ ID NO:38; The polypeptide of claim 10.

12. The spacer domain is a) the CD8 hinge domain, or b) the amino acid sequence of SEQ ID NO:44 or an amino acid sequence that is at least 80% identical to SEQ ID NO:44 The polypeptide of claim 10, comprising:

13. 13. The CAR polypeptide of any one of claims 1 to 12, comprising an amino acid sequence of one of SEQ ID NOs: 80 to 114 or an amino acid sequence that is at least 80% identical to one of SEQ ID NOs: 80 to 114.

14. A polynucleotide encoding the polypeptide of any one of claims 1 to 13.

15. A vector comprising the polynucleotide of claim 14.

16. A lentivirus comprising a polypeptide according to any one of claims 1 to 13, a polynucleotide according to claim 14, or a vector according to claim 15.

17. A cell or a population thereof comprising a polypeptide according to any one of claims 1 to 13, a polynucleotide according to claim 14, a vector according to claim 15, or a lentivirus according to claim 16.

18. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 13, a polynucleotide according to claim 14, a vector according to claim 15, a lentivirus according to claim 16, or a cell or population thereof according to claim 17, and a pharma- ceutically acceptable carrier.

19. A method for increasing activation of NK cells or a population thereof, comprising contacting said NK cells or a population thereof with a polypeptide according to any one of claims 1 to 13, a polynucleotide according to claim 14, a vector according to claim 15, or a lentivirus according to claim 16.

20. Use of a polypeptide according to any one of claims 1 to 13, a polynucleotide according to claim 14, a vector according to claim 15, a lentivirus according to claim 16, a cell or population thereof according to claim 17, or a pharmaceutical composition according to claim 18 in the manufacture of a CAR-based therapeutic.

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